Production of insulin producing cells

ABSTRACT

The invention provides methods for differentiating stem cells along the pancreatic lineage as well as large scale culture methods. The present invention further provides pancreatic progenitor cells derived from stem cells to provide pancreatic cells to a subject.

RELATED APPLICATION

This application claims priority from U.S. Provisional Application Ser.No. 61/355,916 filed Jun. 17, 2010, the contents of which areincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION Pancreas

The pancreas is an elongated, tapered organ which lies to the rear ofthe upper left hand side of the abdominal cavity. It has beenanatomically described as containing three main sections including ahead (widest end—located near the duodenum), a body, and a tail (taperedend—located near the spleen). This organ houses two main tissue types:exocrine tissue, comprised of both acinar and ductal cells; andendocrine tissue, containing cells which produce hormones (i.e.,insulin) for delivery into the bloodstream. The exocrine pancreas,comprising about 95% of the pancreatic mass, is an acinar glandcontaining clusters of pyramidal secretory cells (referred to as acini)that produce digestive enzymes (i.e., amylase, lipase, phospholipase,trypsin, chymotrypsin, aminopeptidase, elastase and various otherproteins). These enzymes are delivered to the digestive system by tubesconstructed of cuboidal ductal cells, which also produce bicarbonate fordigestive purposes. Between the secretory acini and ductal tubes islocated a connecting cell component referred to as centroacinar cells.

The endocrine pancreas, comprising only about 1-2% of the pancreaticmass, contains clusters of hormone-producing cells referred to as isletsof Langerhans (the islet cells are responsible for the maintenance ofblood glucose levels by secreting insulin). These clusters are made upof at least seven cell types, including, but not limited to,insulin-producing β-cells, glucagon-producing α-cells,somatostatin-producing δ-cells, and PP-cells which produce pancreaticpolypeptide. In addition, a subpopulation of endocrine cells referred toas ε-cells has been described.

Diabetes

Diabetes mellitus is a medical condition characterized by variable yetpersistent high blood-glucose levels (hyperglycemia). Diabetes is aserious devastating illness that is reaching epidemic proportions inboth industrialized and developing countries. In 1985, there wereapproximately 30 million people with diabetes worldwide, which increased135 million in 1995 and is expected to increase further by close to 50%by 2050. Diabetes is the fifth leading cause of death in the UnitedStates. According to the American Diabetes Association, the economiccost of diabetes in the U.S. in 2002 was $132 billion, including $92billion of direct costs. This figure is expected to reach in excess of$190 billion by 2020.

Generally, diabetes mellitus can be subdivided into two distinct types:Type 1 diabetes and Type 2 diabetes. Type 1 diabetes is characterized bylittle or no circulating insulin and it most commonly appears inchildhood or early adolescence. It is caused by the destruction of theinsulin-producing beta cells of the pancreatic islets. To survive,people with Type 1 diabetes must take multiple insulin injections dailyand test their blood sugar multiple times per day. However, the multipledaily injections of insulin do not adequately mimic the body'sminute-to-minute production of insulin and precise control of glucosemetabolism. Blood sugar levels are usually higher than normal, causingcomplications that include blindness, renal failure, non-healingperipheral vascular ulcers, the premature development of heart diseaseor stroke, gangrene and amputation, nerve damage, impotence and itdecreases the sufferer's overall life expectancy by one to two decades.

Type 2 diabetes usually appears in middle age or later and particularlyaffects those who are overweight. In Type 2 diabetes, the body's cellsthat normally require insulin lose their sensitivity and fail to respondto insulin normally. This insulin resistance may be overcome for manyyears by extra insulin production by the pancreatic beta cells.Eventually, however, the beta cells are gradually exhausted because theyhave to produce large amounts of excess insulin due to the elevatedblood glucose levels. Ultimately, the overworked beta cells die andinsulin secretion fails, bringing with it a concomitant rise in bloodglucose to sufficient levels that it can only be controlled by exogenousinsulin injections. High blood pressure and abnormal cholesterol levelsusually accompany Type 2 diabetes. These conditions, together with highblood sugar, increase the risk of heart attack, stroke, and circulatoryblockages in the legs leading to amputation.

There is a third type of diabetes in which diabetes is caused by agenetic defect, such as Maturity Onset Diabetes of the Young (MODY).MODY is due to a genetic error in the insulin-producing cells thatrestricts its ability to process the glucose that enters this cell via aspecial glucose receptor. Beta cells in patients with MODY cannotproduce insulin correctly in response to glucose, resulting inhyperglycemia and require treatment that eventually also requiresinsulin injections.

The currently available medical treatments for insulin-dependentdiabetes are limited to insulin administration, pancreas transplantation(either with whole pancreas or pancreas segments) and pancreatic islettransplantation. Insulin therapy is by far more prevalent than pancreastransplantation and pancreatic islet transplantation. However,controlling blood sugar is not simple. Despite rigorous attention tomaintaining a healthy diet, exercise regimen, and always injecting theproper amount of insulin, many other factors can adversely affect aperson's blood-sugar control including: stress, hormonal changes,periods of growth, illness or infection and fatigue. People withdiabetes must constantly be prepared for life threatening hypoglycemic(low blood sugar) and hyperglycemic (high blood sugar) reactions.

In contrast to insulin administration, whole pancreas transplantation ortransplantation of segments of the pancreas is known to have cureddiabetes in patients. However, due to the requirement for life-longimmunosuppressive therapy, the transplantation is usually performed onlywhen kidney transplantation is required, making pancreas-onlytransplantations relatively infrequent operations. Although pancreastransplants are very successful in helping people with insulin-dependentdiabetes improve their blood sugar to the point they no longer needinsulin injections and reduce long-term complications, there are anumber of drawbacks to whole pancreas transplants. Most importantly,getting a pancreas transplant involves a major operation and requiresthe use of life-long immunosuppressant drugs to prevent the body'simmune system from destroying the pancreas that is a foreign graft.Without these drugs, the pancreas is destroyed in a matter of days. Therisks in taking these immunosuppressive drugs is the increased incidenceof infections and tumors that can both be life threatening.

Pancreatic islet transplants are much simpler and safer procedures thanwhole pancreas transplants and can achieve the same effect by replacingbeta cells. However, the shortage of islet cells available fortransplantation remains an unsolved problem in islet celltransplantation. Since islets form only about 2% of the entire pancreas,isolating them from the rest of the pancreas that does not produceinsulin takes approximately 6 hours. Although an automated isolationmethod has made it possible to isolate enough islets from one pancreasto transplant into one patient, as opposed to the 5 or 6 organspreviously needed to carry out one transplant, the demand for isletsstill exceeds the currently available supply of organs harvested fromcadavers. Additionally, long term resolution of diabetic symptoms isoften not achieved.

Stem Cells

Pluripotent stem cells including embryonic stem (ES) cells (Evans andKaufman (1981); Martin (1981); Thomson et al. (1998)) and inducedpluripotent stem (iPS) cells (Takahashi and Yamanaka (2006); Takahashiet al. (2007); Yu et al. (2007)) can be infinitely expanded in vitro anddifferentiated into any cell type when exposed to the appropriatesignals (Keller et al. (2005); Soria et al. (2001); Kumar et al. (2003);Magliocca and Odorico (2006); Madsen (2006)). Previous studies haveshown that human ES cells can be directed to differentiate intofunctional endocrine cells, and that transplantation of thesepancreatic-like cells derived from human ES (hES) cells in vitronormalizes glucose levels in diabetic mice (Shim et al. (2007); Jiang etal. (2007); Philips et al. (2007); D'Amour et al. (2005); D'Amour et al.(2005); Kroon et al. (2008)). Induced pluripotent stem cells have beengenerated from non-diabetic and diabetic donors, and induced todifferentiate into pancreatic insulin-producing cells, although nodemonstration of function in vivo have been reported (Zhang et al.(2009); Tateishi et al. (2008); Maehra et al. (2009)). Inducedpluripotent stem cells have the advantage of being accessible from anyindividual, and thus, could provide patient-specific donor cell sourcefor a range of diseases. Prior studies of directed differentiation ofpluripotent cells to insulin-producing cells required multi-step cultureprocedures using multiple cytokines.

SUMMARY OF THE INVENTION

Described herein is a differentiation method that provides reproduciblepatterns of differentiation to pancreatic cells from multiple human ESand iPS lines capable of reversing diabetes in mice using only fouradded proteins. Additionally, it is demonstrated herein that thesuspension differentiation strategy can be scaled from about 1 mLvolumes in static culture to about 100 mL and higher volumes (including,but not limited to, about 10 ml, about 20 ml, about 30 ml, about 40 ml,about 50 ml, about 60 ml, about 70 ml, about 80 ml, about 90 ml, about100 ml, about 150 ml, about 200 ml, about 250 ml, about 300 ml, about350 ml, about 400 ml, about 450 ml, about 500 ml, about 550 ml, about600 ml, about 650 ml, about 700 ml, about 750 ml, about 800 ml, about850 ml, about 900 ml, about 950 ml, about 1 liter, about 1.5 liters,about 2 liters about 2.5 liters, about 3 liters, about 3.5 liters, about4 liters, about 4.5 liters, about 5 liters, about 5.5 liters, about 6liters, about 6.5 liters, about 7 liters, about 7.5 liters, about 8liters, about 8.5 liters, about 9 liters, about 9.5 liters, about 10liters, about 15 liters, about 20 liters, about 25 liters, about 30liters, about 35 liters, about 40 liters, about 45 liters, about 50liters, about 100 liters, about 200 liters, about 300 liters, about 400liters, about 500 liters and higher) in stirred bioreactors. Thus, thegrowth of cells in large numbers to facilitate transplantation in largeanimal and human transplantation has been achieved. The results indicatethat the efficiency of iPS cell differentiation into insulin-producingcells was comparable in a stirred suspension bioreactor culture withstatic culture. The bioreactor allowed the culture of cells at higherdensities, without loss of cells or viability. Diabetic mice wererescued by transplantation of iPS-derived cells from 100 ml bioreactorcultures. This is the first demonstration of large-scale culture ofpancreatic islet cells suitable for transplantation therapies.

One embodiment provides compositions and methods for providinginsulin-expressing cells and progenitors from stem and iPS cells.

One embodiment provides a method to differentiate stem cells topancreatic progenitor cells comprising the steps of: a) contacting thestem cells with at least one member of the TGFβ family of cytokines andat least one member of the Wnt family of proteins, b) contacting thecells obtained from step a) with at least one member of the TGFβ familyof cytokines, at least one member of the Wnt family of proteins, and anagent that inhibits sonic hedgehog activity; and c) contacting the cellsobtained from step b) with a member of the Epidermal growth factor (EGF)family of proteins; so as to yield pancreatic progenitor cells. In oneembodiment, the at least one member of the TGFβ family of cytokines isactivinA or nodal. In another embodiment, the at least one member of theWnt family is Wnt3 or Wnt3A. In another embodiment, the at least onemember of the EGF family is EGF.

Another embodiment provides a method to differentiate stem cells topancreatic progenitor cells comprising the steps of: a) contacting theiPS cells with Activin A and Wnt3a; b) contacting the cells obtainedfrom step a) with Activin-A, Wnt3a, and an agent that inhibits sonichedgehog activity; and c) contacting the cells obtained from step b)with EGF; so as to yield pancreatic progenitor cells. One embodimentprovides the a method to differentiate stem cells to pancreaticprogenitor cells consisting essentially of the steps of: a) contactingthe iPS cells with Activin A and Wnt3a; b) contacting the cells obtainedfrom step a) with Activin-A, Wnt3a, and an agent that inhibits sonichedgehog activity; and c) contacting the cells obtained from step b)with EGF; so as to yield pancreatic progenitor cells.

In one embodiment, the stem cells are embryonic (embryonic stem (ES)cell has unlimited self-renewal and can differentiate into all tissuetypes; ES cells are derived from the inner cell mass of the blastocystor primordial germ cells from a post-implantation embryo (embryonic germcells or EG cells)) or adult stem cells (e.g., MAPCs or MIAMI(marrow-isolated adult multilineage inducible) cells). In anotherembodiment, the stem cells are induced pluripotent stem (iPS) cells. Inone embodiment, the stem cells are mammalian cells, such as human cells.

One embodiment further provides contacting the cells obtained from stepc) with at least one member of the TGFβ family of cytokines, at leastone member of the Wnt family of proteins, exendin4 or a combinationthereof to yield cells expressing insulin. In one embodiment, the atleast one member of the TGFβ family of cytokines is GDF-11. In anotherembodiment, the at least one member of the Wnt family of proteins isbetacellulin.

One embodiment provides for contacting the cells obtained from step c)with GDF-11, betacellulin, exendin4 or a combination thereof to yieldcells expressing insulin. In one embodiment, the cells expressinginsulin or having increased expression of insulin secrete insulin,c-peptide or a combination thereof. In one embodiment, the insulin isinsulin-1.

In one embodiment, the agent that inhibits sonic hedgehog activity iscyclopamine or an anti-SHH antibody.

In one embodiment, the differentiation of the stem cell occurs in a cellculture dish. In another embodiment, the differentiation of the stemcell occurs in a bioreactor.

One embodiment provides a composition comprising or consistingessentially of Activin-A, Wnt 3a and an agent that inhibits sonichedgehog activity and stem cells. In one embodiment, the compositioncomprises the cells prepared by methods described herein and cellculture medium or a pharmaceutically acceptable carrier. One embodimentprovides a method to prepare a composition comprising combining cellsobtained by the methods described herein of with cell culture medium ora pharmaceutically acceptable carrier.

One embodiment provides a method to provide pancreatic cells to asubject in need thereof comprising: a) contacting the stem cells with atleast one member of the TGFβ family of cytokines and at least one memberof the Wnt family of proteins, b) contacting the cells obtained fromstep a) with at least one member of the TGFβ family of cytokines, atleast one member of the Wnt family of proteins, and an agent thatinhibits sonic hedgehog activity; and c) contacting the cells obtainedfrom step b) with a member of the Epidermal growth factor (EGF) familyof proteins; and administering the cells so as to provide pancreaticcells in the subject. In one embodiment, the at least one member of theTGFβ family of cytokines is activinA or nodal. In another embodiment,the at least one member of the Wnt family is Wnt3 or Wnt3A. In anotherembodiment, the at least one member of the EGF family is EGF.

One embodiment provides a method to provide pancreatic cells to asubject in need thereof comprising: a) contacting stem cells withActivin A and Wnt 3a; b) contacting the cells obtained from step a) withActivin-A, Wnt3a, and an agent that inhibits sonic hedgehog activity; c)contacting the cells obtained from step b) with EGF; and administeringthe cells so as to provide pancreatic cells in the subject. In oneembodiment, the stem cells are embryonic or adult stem cells (e.g.,MAPCs). In another embodiment, the stem cells are induced pluripotentstem (iPS) cells.

In one embodiment, the obtained in step c) are contacted with at leastone member of the TGFβ family of cytokines, at least one member of theWnt family of proteins, exendin4 or a combination thereof to yield cellsexpressing insulin prior to administration to the subject. In oneembodiment, the least one member of the TGFβ family of cytokines isGDF-11. In another embodiment, the at least one member of the Wnt familyor proteins is betacellulin.

In one embodiment, the cells obtained from step c) are contacted withGDF-11, betacellulin, exendin4 or a combination thereof to yield cellsexpressing insulin prior to administration to the subject.

Another embodiment provides a method to provide insulin expressing cellsto a subject in need thereof comprising: a) contacting stem cells withActivin A and Wnt 3a; b) contacting the cells obtained from step a) withActivin-A, Wnt3a, and an agent that inhibits sonic hedgehog activity; c)contacting the cells obtained from step b) with EGF; d) contacting thecells obtained from step c) with GDF-11, betacellulin, exendin4 or acombination thereof so as to yield cells expressing insulin or havingincreased expression of insulin; and e) administering the cellsexpressing insulin or having increased expression of insulin to thesubject. In one embodiment, the stem cells are embryonic or adult stemcells (e.g., MAPCs or MIAMI cells). In another embodiment, the stemcells are induced pluripotent stem (iPS) cells.

In one embodiment, the subject is a mammal, such as a human. In oneembodiment, the subject has a pancreatic disorder or injury, such asdiabetes (e.g., Type I or Type II diabetes), obesity, pancreaticatresia, pancreas inflammation, alpha1-antitrypsin deficiency,hereditary pancreatitis, pancreatic cancer, pancreatic enzymedeficiency, hyperinsulinism, physical trauma, chemical, radiation,aging, disease or combination thereof.

One embodiment provides for the use of cells prepared by the methodsdescribed herein to prepare a medicament to treat a pancreatic disorderor injury, such as pancreatic disorder comprises diabetes (e.g., Type Ior Type II diabetes), obesity, pancreatic atresia, pancreasinflammation, alpha1-antitrypsin deficiency, hereditary pancreatitis,pancreatic cancer, pancreatic enzyme deficiency, hyperinsulinism,physical trauma, chemical, radiation, aging, disease or combinationthereof. In one embodiment, the medicament further comprises aphysiologically acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic representation of the procedure for directeddifferentiation of human iPS cells in small static culture and stirredbioreactor cultures in vitro, and transplantation in vivo.

FIGS. 2A-D. Directed differentiation of iPS cells into insulin-producingcells progresses through developmental stages. L-1 iPS cells weredirected to differentiate in EB suspension culture for 21 days withsequential cytokine stimulation to support differentiation intoinsulin-producing cells. Quantitative PCR analysis indicated that: (a)iPS cells differentiated into definitive endoderm, followed bypancreatic endoderm and (b) maturation of endocrine cells, in whichinsulin and related genes Rfx6 and glucokinase, markers of mature betacells can be found in EB cultures. (c) Immunostaining of specificpancreatic marker proteins (pdx-1, insulin) in iPS derived cells atstage 4. (d) C-peptide secreted by iPS-derived cells was measured in24-hour conditioned medium from undifferentiated (day 0), day 15, andday 21 cultured cells. C-peptide was measured by ELISA assay, and isused to estimate the amount of insulin secretion.

FIGS. 3A-B. Transplantation of induced L-1 iPS cells partiallyameliorates diabetic symptoms. Changes in (a) blood glucose levels and(b) body weight were monitored daily in STZ-treated diabetic micetransplanted under the kidney capsule with cells differentiated for 9,15 and 21 days. The solid vertical line indicates the time of graftremoval. Mice were monitored for an additional 7 days to observe glucoselevels and weight loss. After graft explant, weight loss was observed,suggesting that grafted iPS derived cells improved glycemic control.

FIGS. 4A-B. Differentiated iPS cells reverse diabetes in mice. Day 15differentiated iPS cultures (3-8×10⁶ cells) were placed under the kidneycapsule (K) or in the epididymal fat pad (EFP) of STZ-induced diabeticnude mice (n=6). After transplantation, daily insulin injections wereadministered until blood glucose levels were below 350 mg/dL. Allglucose levels are non-fasting. The blood glucose level comparisonbetween the pre (from day −40 to day 0) and post transplantation (fromday 0 to day 56) period for each mouse. The shaded area shows theaverage normal blood glucose level plus and minus two standardderivations in the mice.

FIGS. 5A-B. iPS-derived grafts contain insulin-producing cells. Graftswere removed from mice 56 days after transplantation for insulinexpression analysis. Grafts under the kidney capsule (g) were separatedfrom kidney tissue (k) and grafts in the epididymal fat pad (e) wereanalyzed intact. (a) Quantitative RT-PCR was performed for insulin andRfx6 expression in excised grafts. G, GAPDH; I, Insulin; R, Rfx6; hfp,human fetal pancreas. (b) Grafts were sectioned and stained for humanpro-insulin and pdx-1, markers of mature beta cells. Clusters of cellsco-expressing pdx-1 and pro-insulin are present in the grafts inaddition to pro-insulin expressing cells that are negative for pdx-1.

FIG. 6. Directed differentiation of human H9 ES cells in vitroprogresses through developmental stages. Quantitative PCR analysis ofthe definitive endoderm (Foxa2), pancreatic endoderm (Pdx-1, Ptf1a) andendocrine (Insulin) markers during differentiation of human H9 embryonicstem cells demonstrated sequential stages of pancreatic development.Relative levels of gene expression were normalized to the GAPDH mRNAlevels. The value of undifferentiated (day 0) H9 cells is set at 1.

FIGS. 7A-B. The iPS cell line L-1 is pluripotent. (a) Immunofluorescenceanalysis of L-1 and H9 cells demonstrates that morphology and expressionof several hES cell markers are similar in the two cell lines. (b) Geneexpression patterns were compared between parental fibroblasts (NHDF),two iPS lines derived from NHDF cells (L1, L2) and the hES H9 cell line.Genes specific for pluripotent stem cells were expressed in common foriPS and ES cells, and differ from NHDF parental cells.

FIG. 8. The iPS cell line L-1 is pluripotent. L-1 cells weredifferentiated in suspension culture by EB (embryoid body) formation fortwo weeks. Quantitative PCR was performed, demonstrating that the L-1cells differentiated into representative tissues of the three germlayers: Oct4, undifferentiated pluripotent cells; VE Cadherin, mesoderm;NCAM, ectoderm; AFP, endoderm.

FIGS. 9A-G: Comparison of EB growth in small static and stirredbioreactor culture. (a) EB morphology at different time points ofdifferentiation. (b) EB growth detected by cell density and viability.(c), (d), (e) EB growth determined by measurement of EB number and EBsize. (f), (g) total DNA and protein amount static and stirredbioreactor culture were compared as an estimate of cell numbers.

FIGS. 10A-B: Directed differentiation of iPS cells in stirredbioreactors exhibited similar development patterns as small-scale staticcultures. (a) Comparison of each stage gene expression was performed byqPCR. Gene expression in individual samples were normalized to the GAPDHgene expression. The value of undifferentiated (d0) iPS cells was setas 1. (b) Summary of definitive endoderm and pancreatic endoderm geneexpression in multiple bioreactor and static cultures. Gene expressionfrom static cultures was set as 1 for each time point.

FIGS. 11A-C: Human iPS cells differentiated in stirred bioreactorcultures reversed diabetes in mice. (a) Day 15 differentiated iPScultures from 10 ml static culture (group 1) were placed under thekidney capsule and (b) day 15 differentiated iPS culture from 100 mlstirred bioreactor culture (group 2&3) were placed in kidney capsule orthe epididymal fat pad (EFP) of diabetic nude mice. Non-fasting bloodglucose levels were monitored daily. The shaded area shows the averageblood glucose level +/− two standard derivations form non-diabetic nudemice. (c) Comparison of average glucose levels for each mouse aftertransplantation, 10 ml static cells transplanted mice required insulinadministration throughout the post-transplant period. Mice transplantedwith 100 ml stirred bioreactor cells demonstrated a decrease in bloodglucose levels controlled by the engrafted cells. The line indicated thesurvival glucose level (350 μg/ml).

DETAILED DESCRIPTION OF THE INVENTION

Islet transplantation is a promising treatment for diabetes, such astype 1 diabetes, but this application is limited by the availability ofdonor tissues. Human pluripotent stem cells, including embryonic stem(ES) cells and induced pluripotent stem (iPS) cells have potential todifferentiate into virtually all cell types, and several groups havedemonstrated that hES and iPS cells differentiate into insulin-producingcells. Herein the derivation of human iPS cells from neonatal foreskinfibroblast cells is described in which the cells were reprogrammed by acombination transduction with human Oct4, Sox3, Nanog and Lin28 genes.One of the iPS cell lines, L-1 was characterized by karyotyping,immunostaining and multilineage differentiation by embryoid body (EB)formation, and expressed human pluripotent stem cell-specific genes suchas Oct4, Tra-1-81 and SSEQ-4. Using a four step differentiation process,insulin producing cells were generated. The differentiation todefinitive endoderm was identified first based on the expression ofspecific definitive endoderm markers FoxA2 and Sox17 using qPCR. As manyas fifty percent of cells in the first differentiation stage were CXCR4and E-Cad double positive cells by flow cytometric analysis (FACS),which was higher than human ES differentiation cultures in the sameconditions. Pancreatic endoderm was identified by high levels ofexpression of specific pancreatic progenitor markers Pdx-1 and Ptf1.Finally, after expansion of pancreatic endoderm, insulin producing cellswere generated by the addition of maturation factors. Insulin expressionwas detected by q-PCR and c-peptide release was confirmed byimmunostaining. C peptide secreted in supernatant was also detected byELISA. Thus, insulin producing cells can be generated efficiently fromiPS cells in vitro. Further, these cells are capable of reversingdiabetes in vivo.

The development of large-scale culture methods for effective celldifferentiation and expansion would aid in the achievement of the goalof providing a source of functional cells for cell replacementtherapies. Also described herein below is the amplification of human iPScell differentiation by embryoid body (EB) formation, from small-scalestatic cultures to dynamic stirred bioreactor cultures. Static andstirred-bioreactor methods were compared by growth rate, cell viability,differentiation efficiency to pancreatic progenitor cells and functionanalysis in vivo after transplantation into diabetic mice. The resultsindicated that the bioreactor culture provided 10-100-fold increase inculture volume and cell numbers, with similar in vitro parameters asstatic cultures, as well as an efficient differentiation scheme. TheiPS-derived pancreatic progenitor cells from stirred bioreactors alsoenhanced survival of diabetic mice by maintaining body weight and bydecreasing glucose levels in transplanted mice, resulting innormoglycemia in a subset of mice. The stirred bioreactor chambers canbe increased in size to further increase the volume and cell yield.Thus, a directed differentiation protocol for human iPS cells intopancreatic cells can be achieved efficiently in large-scale culture.This system provides a method for producing unlimited allogeneic orpatient-specific functional cells for the treatment of diabetes andother diseases.

DEFINITIONS

As used herein, the terms below are defined by the following meanings:“Expansion” refers to the propagation of cells without differentiation,including the proliferation of any cell type without significant furtherdifferentiation.

“Progenitor cells” are cells produced during differentiation of a stemcell that have some, but not all, of the characteristics of theirterminally-differentiated progeny. Defined progenitor cells, such as“pancreatic progenitor cells,” are committed to a lineage, but not to aspecific or terminally-differentiated cell type.

An effective amount of an agent (e.g., Activin-A, an agent that inhibitsSHH, EGF, Wnt3a, exendin4, GDF11 or betacellulin) is an amount effectiveto differentiate the cells as recited, when applied alone or incombination with one or more other agents.

“Increased expression” of a marker (e.g., Foxa2, Sox17, CXCR4, ECad,Pdx-1, Ptf1a, Rfx6, Gluck, Insulin) refers to an increase (in mRNAand/or protein) relative to the parent cell (a cell prior to the recitedtreatment (e.g., contacting with Activin-A etc.) and/or treatments) onan average per cell basis (for example, if the parent cell does notexpress a marker and the progeny does, there is an increase inexpression; or if the progeny expresses more of the marker compared tothe parent cell there is also an increase in expression).

“Engraft” or “engraftment” refers to the process of cellular contact andincorporation into an existing tissue or site of interest. In oneembodiment, greater than about 5%, greater than about 10%, greater thanabout 15%, greater than about 20%, greater than about 25%, greater thanabout 30%, greater than about 35%, greater than about 40%, greater thanabout 45%, greater than about 50%, greater than about 55%, greater thanabout 60%, greater than about 65%, greater than about 70%, greater thanabout 75%, greater than about 80%, greater than about 85%, greater thanabout 90%, greater than about 95% or about 100% of administered cellsengraft in the pancreas or other tissues.

Persistence refers to the ability of cells to resist rejection andremain or increase in number over time (e.g., days, weeks, months,years) in vivo. Thus, by persisting, the cells can populate the pancreasor other tissues or remain in vivo, such as in barrier devices or otherencapsulated forms.

The term “isolated” refers to a cell or cells which are not associatedwith one or more cells or one or more cellular components that areassociated with the cell or cells in vivo. An “enriched population”refers to a relative increase in numbers of the cell of interestrelative to one or more other cell types in vivo or in primary culture.

A “subject” or cell source can be a vertebrate, including a mammal, suchas a human. Mammals include, but are not limited to, humans, farmanimals, sport animals and companion animals. In included in the term“animal” is dog, cat, fish, gerbil, guinea pig, hamster, horse, rabbit,swine, mouse, monkey (e.g., ape, gorilla, chimpanzee, orangutan) rat,sheep, goat, cow and bird.

As used herein, “treat,” “treating” or “treatment” includes treating,reversing, ameliorating, or inhibiting an injury or disease-relatedcondition or a symptom of an injury or disease-related condition.Prevention of an injury or disease-related condition or a symptom of aninjury or disease-related condition is also carried out by the methodsdescribed herein.

An “effective amount” generally means an amount which provides thedesired effect. For example, an effective dose is an amount sufficientto affect a beneficial or desired result, including a clinical result.The dose can be administered in one or more administrations and caninclude any preselected amount of cells. The precise determination ofwhat would be considered an effective dose may be based on factorsindividual to each subject, including size, age, injury or disease beingtreated and amount of time since the injury occurred or the diseasebegan. One skilled in the art, particularly a physician, would be ableto determine the number of cells that would constitute an effectivedose. Doses can vary depending on the mode of administration, e.g.,local or systemic; free or encapsulated. The effect can be engraftmentor other clinical endpoints, such as reversal or treatment of diabetes.Other effects can include providing beta cells, recruiting endogenouscells, effecting angiogenesis, and/or providing pancreatic progenitors.

“Co-administer” can include sequential, simultaneous and/or separateadministration of two or more agents.

The terms “comprises”, “comprising”, and the like can have the meaningascribed to them in U.S. Patent Law and can mean “includes”, “including”and the like. As used herein, “including” or “includes” or the likemeans including, without limitation.

Stem Cells/iPS Cells

The embryonic stem (ES) cell has unlimited self-renewal and candifferentiate into all tissue types. ES cells are derived from the innercell mass of the blastocyst or primordial germ cells from apost-implantation embryo (embryonic germ cells or EG cells). ES (and EG)cells can be identified by positive staining with antibodies to SSEA 1(mouse) and SSEA 4 (human). At the molecular level, ES and EG cellsexpress a number of transcription factors specific for theseundifferentiated cells. These include Oct-4 and rex-1. Rex expressiondepends on Oct-4. Also found are LIF-R (in mouse) and the transcriptionfactors sox-2 and rex-1. Rex-1 and sox-2 are also expressed in non-EScells. Another hallmark of ES cells is the presence of telomerase, whichprovides these cells with an unlimited self-renewal potential in vitro.

Adult stem cells, such as “Multipotent adult progenitor cells” (MAPCs)are non-embryonic (non-ES), non-germ and non-embryonic germ (non-EG)cells that can differentiate into one or more ectodermal, endodermal andmesodermal cells types. MAPCs can be positive for telomerase, Oct-3A(Oct-3/4) or a combination thereof. MAPCs have the ability to regenerateall primitive germ layers (endodermal, mesodermal and ectodermal) invitro and in vivo. In this context they are equivalent to embryonic stemcells and distinct from mesenchymal stem cells. The biological potencyof MAPCs has been proven in various animal models, including mouse, rat,and xenogeneic engraftment of human stem cells in rats or NOD/SCID mice(Jiang, Y. et al. 2002). Clonal potency of this cell population has beenshown. MAPCs are capable of extensive culture without loss ofdifferentiation potential and show efficient, long term, engraftment anddifferentiation along multiple developmental lineages without evidenceof teratoma formation.

Induced pluripotent stem cell (iPSC) technology is the process ofconverting an adult specialized cell, such as a skin cell, into a stemcell, a process known as dedifferentiation. Nuclear reprogramming, theprocess of converting one cell type into another by resetting thepattern of gene expression, can be achieved through forced expression ofdefined transcription factors. One example is the induced pluripotentstem cells (iPSCs), which can be prepared by transducing up to fourgenes (e.g., Oct4, Sox2, Klf4 and c-Myc, called OSKM hereafter) intodifferentiated somatic cells, such as skin fibroblasts. Other geneswhich can be transduced in place of or in addition to, so as to generateiPS cells, include, for example nanog and lin28

The first mouse iPS cell line was generated in 2006 (Takahashi, 2006),which showed ES-like characteristics in self-renewal, teratoma andchimera formation and differentiation. In 2007 (Takahashi, 2007; Yu,2007), human iPS cell lines were successfully established, which madethe derivation of individual pluripotent stem cells possible from asmall skin biopsy. These and many subsequent studies have confirmedhuman iPS cells are characteristically very similar to ES cells. iPScells can self-renew and are able to maintain an undifferentiated statewhen grown under appropriate conditions. As pluripotent cells, they canalso differentiate into any cell type, including pancreatic cells, whenexposed to environment permissive for, or directing differentiation.Human iPS cell lines have been generated from normal human skin cellsand diabetic donors, all of which had the potential to differentiateinto insulin-producing cells (Zhang, 2009; Tateishi, 2008; Maehr, 2009).

iPS cells can also have therapeutic uses for the treatment of diseasewithout the need for stem cells derived from an embryonic source. Forexample, iPSCs can be created from human patients and can bedifferentiated into many tissues to provide new materials for autologoustransplantation, which can avoid immune rejection of the transplantedtissues. For example, pancreatic beta cells differentiated from apatient's iPSCs can be transplanted into the original patient to treatdiabetes. However, before these derivatives can be used in clinic,procedures must be developed to generate large numbers of functionalcells for preclinical and human trials.

Culture of Cells

Thus, cells or their progeny can be maintained and expanded in culturemedium that is available to the art. Such media include, but are notlimited to, Dulbecco's Modified Eagle's Medium® (DMEM), DMEM F12Medium®, Eagle's Minimum Essential Medium®, F-12K Medium®, Iscove'sModified Dulbecco's Medium®, RPMI-1640 Medium®. Many media are alsoavailable as a low-glucose formulation, with or without sodium pyruvate.

Also contemplated is supplementation of cell culture medium withmammalian sera. Sera often contain cellular factors and components thatare necessary for viability and expansion. Examples of sera includefetal bovine serum (FBS), bovine serum (BS), calf serum (CS), fetal calfserum (FCS), newborn calf serum (NCS), goat serum (GS), horse serum(HS), human serum, chicken serum, porcine serum, sheep serum, rabbitserum, serum replacements, and bovine embryonic fluid. It is understoodthat sera can be heat-inactivated at about 55-65° C. if deemed necessaryto inactivate components of the complement cascade.

Additional supplements can also be used advantageously to supply thecells with the trace elements for optimal growth and expansion. Suchsupplements include insulin, transferrin, sodium selenium andcombinations thereof. These components can be included in a saltsolution such as, but not limited to Hanks' Balanced Salt Solution®(HBSS), Earle's Salt Solution®, antioxidant supplements, MCDB-201®supplements, phosphate buffered saline (PBS), ascorbic acid and ascorbicacid-2-phosphate, as well as additional amino acids. Many cell culturemedia already contain amino acids; however some require supplementationprior to culturing cells. Such amino acids include, but are not limitedto, L-alanine, L-arginine, L-aspartic acid, L-asparagine, L-cysteine,L-cystine, L-glutamic acid, L-glutamine, L-glycine, L-histidine,L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine,L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine and L-valine.It is well within the skill of one in the art to determine the properconcentrations of these supplements.

Antibiotics are also typically used in cell culture to mitigatebacterial, mycoplasmal and fungal contamination. Typically, antibioticsor anti-mycotic compounds used are mixtures of penicillin/streptomycin,but can also include, but are not limited to, amphotericin (Fungizone®),ampicillin, gentamicin, bleomycin, hygromycin, kanamycin, mitomycin,mycophenolic acid, nalidixic acid, neomycin, nystatin, paromomycin,polymyxin, puromycin, rifampicin, spectinomycin, tetracycline, tylosinand zeocin. Antibiotic and antimycotic additives can be of some concern,depending on the type of work being performed. One possible situationthat can arise is an antibiotic-containing media wherein bacteria arestill present in the culture, but the action of the antibiotic performsa bacteriostatic rather than bacteriocidal mechanism. Also, antibioticscan interfere with the metabolism of some cell types.

Hormones can also be advantageously used in cell culture and include,but are not limited to, D-aldosterone, diethylstilbestrol (DES),dexamethasone, β-estradiol, hydrocortisone, insulin, prolactin,progesterone, somatostatin/human growth hormone (HGH), thyrotropin,thyroxine and L-thyronine.

Lipids and lipid carriers can also be used to supplement cell culturemedia, depending on the type of cell and the fate of the differentiatedcell. Such lipids and carriers can include, but are not limited tocyclodextrin (α, β, γ), cholesterol, linoleic acid conjugated toalbumin, linoleic acid and oleic acid conjugated to albumin,unconjugated linoleic acid, linoleic-oleic-arachidonic acid conjugatedto albumin, oleic acid unconjugated and conjugated to albumin, amongothers.

Also contemplated is the use of feeder cell layers. Feeder cells areused to support the growth of fastidious cultured cells, including stemcells. Feeder cells are normal cells that have been inactivated by, forexample, γ-irradiation or x-irradiation. In culture, the feeder layerserves as a basal layer for other cells and supplies cellular factorswithout further growth or division of their own. Examples of feederlayer cells are typically human diploid lung cells, mouse embryonicfibroblasts, Swiss mouse embryonic fibroblasts, but can be anypost-mitotic cell that is capable of supplying cellular components andfactors that are advantageous in allowing optimal growth, viability andexpansion of stem cells.

Cells in culture can be maintained either in suspension or attached to asolid support, such as extracellular matrix components and synthetic orbiopolymers. Stem cells sometimes need additional factors that encouragetheir attachment to a solid support, such as type I, type II and type IVcollagen, concanavalin A, chondroitin sulfate, fibronectin,“superfibronectin” and fibronectin-like polymers, gelatin, laminin,poly-D and poly-L-lysine, thrombospondin and vitronectin.

The maintenance conditions of stem cells can also contain cellularfactors that allow stem cells to remain in an undifferentiated form. Itis advantageous under conditions where the cell must remain in anundifferentiated state of self-renewal for the medium to contain forexample epidermal growth factor (EGF), platelet derived growth factor(PDGF), FGF (such as bFGF (FGF-2), leukemia inhibitory factor (LIF; inselected species), and combinations thereof. It is apparent to thoseskilled in the art that supplements that allow the cell to self-renewbut not differentiate should be removed from the culture medium prior todifferentiation.

Stem cell lines and other cells can benefit from co-culturing withanother cell type. Such co-culturing methods arise from the observationthat certain cells can supply cellular factors that allow the stem cellto differentiate into a specific lineage or cell type. These cellularfactors can also induce (suppress) expression of cell-surface,cytoplasmic and nuclear molecules, which can used as markers and aretherefore readily identified by through various methods, including, suchas by monoclonal antibodies. They may also regulate cellular functions.Generally, cells for co-culturing are selected based on the type oflineage one skilled in the art wishes to induce, and it is within thecapabilities of the skilled artisan to select the appropriate cells forco-culture.

Differentiation of Stem Cells to Pancreatic Cells

Stem/iPs cells and pancreatic progenitor cells differentiated fromstem/iPS cells are useful as a source of pancreatic cells. Thematuration, proliferation and differentiation of stem/iPS cells may beeffected through culturing stem/iPS cells with appropriate factors(examples of nucleotide/protein accession numbers provided) including,but not limited to, activin-A (generally two subunits of NM_(—)002192)or other members TGFβ family of cytokines (e.g., BMP-4), including, butnot limited the nodal subset of the TGFβ family of cytokines (activinand nodal related factors, including, but not limited to, nodal,activina and activinb), Wnt3a (or other members of the Wnt family,including, but not limited to, WNT1 (NM_(—)005430; NM_(—)021279;NP_(—)005421; NP_(—)067254), WNT2 (NM_(—)003391; NM_(—)023653;NP_(—)003382; NP_(—)076142), WNT2B (NM_(—)004185; NM_(—)009520;NP_(—)004176; NP_(—)033546), WNT3 (NM_(—)030753; NM_(—)009521;NP_(—)110380; NP_(—)033547), WNT3A (NM_(—)033131; NM_(—)009522;NP_(—)149122; NP_(—)033548), WNT4 (NM_(—)030761; NM_(—)009523;NP_(—)110388; NP_(—)033549), WNT5A (NM_(—)003392; NM_(—)009524;NP_(—)003383; NP_(—)033550), WNT5B (NM_(—)030775; NM_(—)009525;NP_(—)110402; NP_(—)033551), WNT6 (NM_(—)006522), WNT7A (NM_(—)004625;NM_(—)009527; NP_(—)004616; NP_(—)033553), WNT7B (NM_(—)058238;NM_(—)009528; NP_(—)478679; NP_(—)033554), WNT8A, WNT8B (NM_(—)003393;NM_(—)011720; NP_(—)003384; NP_(—)035850), WNT9A (NM_(—)003395;NM_(—)139298; NP_(—)003386; NP_(—)647459), WNT9B, WNT10A (NM_(—)025216;NM_(—)009518; NP_(—)079492 NP_(—)033544), WNT10B (NM_(—)003394;NM_(—)011718; NP_(—)003385; NP_(—)035848), WNT11 (NP_(—)004617;NP_(—)033545; NP_(—)004617; NP_(—)033545), WNT16 (NM_(—)016087;NM_(—)053116; NP_(—)057171 NP_(—)444346)), an agent that inhibits sonichedgehog activity (including, but not limited to, cyclopamine andanti-SHH antibody), HGF (Hepatocyte growth factor/scatter factor;NM_(—)000601; NM_(—)010427; NP_(—)000592; NP_(—)034557), a member of theEpidermal growth factor (EGF) family (including, but not limited to, EGF(NM_(—)001963; NM_(—)010113; NP_(—)001954; NP_(—)034243),Heparin-binding EGF-like growth factor (HB-EGF; NM_(—)001945;NM_(—)010415; NP_(—)001936; NP_(—)034545), transforming growth factor-α(TGF-α; NM_(—)003236; NM_(—)031199; NP_(—)003227; NP_(—)112476),Amphiregulin (AR; NM_(—)001657; NM_(—)009704; NP_(—)001648;NP_(—)033834), Epigen (NM_(—)001013442), Betacellulin (BTC;NM_(—)001729; NM_(—)007568; NP_(—)001720; NP_(—)031594), neuregulin-1(NRG1; NM_(—)004495; XM_(—)620642; NP_(—)004486; XP_(—)620642),neuregulin-2 (NRG2; XM_(—)001129975; XP_(—)001129975), neuregulin-3(NRG3; NM_(—)001165972; NM_(—)008734; NP_(—)001010848; NP_(—)032760),neuregulin-4 (NRG4; NM_(—)138573; NM_(—)032002; NP_(—)612640;NP_(—)114391), any protein which contains one or more repeats of theconserved amino sequence CX₇CX₄₋₅CX₁₀₋₁₃CXCX₈GXRC, where X representsany amino acid, or a combination thereof), or other mitogenic proteins,exendin (including, but not limited to, exendin 4(H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2;SEQ ID NO:11) and exenatide (a synthetic 39-amino acid peptide whichclosely resembles exendin-4 and is marketed by Amylin Pharmaceuticalsand Eli Lilly and Company as Byetta™ for the treatment of diabetes; CASnumber 141732-76-5)), Growth differentiation factor 11 (GDF11) alsoknown as bone morphogenetic protein 11 (BMP-11) or other members of thebone morphogenetic protein/transforming growth factor beta (BMP/TGFbeta)superfamily, and/or betacellulin (or other members of the EGF family).These proteins can generally be used in the amount of, for example,about 0.5 to about 200 ng/ml or about 5 nM to about 30 nM.

The transforming growth factor beta (TGF-β) family is a large family ofstructurally related cell regulatory proteins((LIVM)-x(2)-P-x(2)-[FY]-x(4)-C-x-G-x-C). Proteins from the TGF-betafamily are generally active as a homo- or heterodimer; the two chainsbeing linked by a disulfide bond. Members of the TGFβ family ofcytokines (with examples of nucleotide/protein accession numbers forthese members) include, but are not limited to, AMH (NM_(—)000479);ARTN; BMP10 (NM_(—)014482; NM_(—)009756; NP_(—)055297; NP_(—)033886);BMP15 (NM_(—)005448; NM_(—)009757; NP_(—)005439; NP_(—)005439); BMP2(NM_(—)001200; NM_(—)007553; NP_(—)001191; NP_(—)031579); BMP3(NM_(—)001201; NM_(—)173404; NP_(—)001192; NP_(—)775580); BMP4(NM_(—)001202; NM_(—)007554; NP_(—)001193; NP_(—)031580); BMP5(NM_(—)021073; NM_(—)007555; NP_(—)066551; NP_(—)031581); BMP6(NM_(—)001718; NM_(—)007556; NP_(—)001709; NP_(—)031582); BMP7(NM_(—)001719; NM_(—)007557; NP_(—)001710; NP_(—)031583); BMP8A(NM_(—)181809; NM_(—)007558; NP_(—)861525; NP_(—)031584); BMP8B(NM_(—)001720; NM_(—)001720); GDF1 (NM_(—)001492; NM_(—)008107;NP_(—)001483; NP_(—)032133); GDF10 (NM_(—)004962; NM_(—)145741;NP_(—)004953; NP_(—)665684); GDF11 (NM_(—)005811; NM_(—)010272;NP_(—)005802; NM_(—)010272); GDF15 (NM_(—)004864; NM_(—)011819;NP_(—)004855; NP_(—)035949); GDF2 (NM_(—)016204; NM_(—)019506;NP_(—)057288; NP_(—)062379); GDF3 (NM_(—)020634; NM_(—)008108;NP_(—)065685; NP_(—)032134); GDF3A; GDF5 (NM_(—)000557; NM_(—)008109;NP_(—)000548; NP_(—)032135); GDF6 (NM_(—)001001557; NM_(—)013526;NP_(—)038554); GDF7 (NM_(—)182828; NM_(—)013527; NP_(—)878248;NP_(—)038555); GDF8 (NM_(—)005259; NM_(—)010834; NP_(—)005250;NP_(—)034964); GDF9 (NM_(—)005260; NM_(—)008110; NP_(—)005251;NP_(—)032136); GDNF (NM_(—)000514; NM_(—)010275; NP_(—)000505;NP_(—)034405); INHA (NM_(—)002191; NM_(—)010564; NP_(—)002182;NP_(—)034694); INHBA (NM_(—)002192; NM_(—)008380; NP_(—)002183;NP_(—)032406); INHBB (NM_(—)002193; XM_(—)984243; NP_(—)002184;XP_(—)989337); INHBC (NM_(—)005538; NM_(—)010565; NP_(—)005529;NP_(—)034695); INHBE; LEFTY1; LEFTY2; MSTN (NM_(—)005259; NM_(—)010834;NP_(—)005250; NP_(—)034964); NODAL (NM_(—)018055; NM_(—)013611;NP_(—)060525; NP_(—)038639); NRTN (NM_(—)004558); PSPN; TGFB1(NM_(—)000660; NM_(—)011577; NP_(—)000651; NP_(—)035707); TGFB2(NM_(—)003238; NM_(—)009367; NP_(—)003229; NP_(—)033393); and TGFB3(NM_(—)003239; XM_(—)994378; NP_(—)003230; XP_(—)999472).

For example, sequences for use in the invention have at least about 50%or about 60% or about 70%, about 71%, about 72%, about 73%, about 74%,about 75%, about 76%, about 77%, about 78%, or about 79%, or at leastabout 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about86%, about 87%, about 88%, or about 89%, or at least about 90%, about91%, about 92%, about 93%, or about 94%, or at least about 95%, about96%, about 97%, about 98%, or about 99% sequence identity compared tothe accession numbers provided herein and/or any other such sequenceavailable to an art worker, using one of alignment programs available inthe art using standard parameters. In one embodiment, the differences insequence are due to conservative amino acid changes. In anotherembodiment, the protein sequence or DNA sequence has at least 80%, atleast 85%, at least 90%, at least 95% sequence identity with thesequences disclosed herein and is bioactive (e.g., retains activity).

Methods of alignment of sequences for comparison are available in theart. Thus, the determination of percent identity between any twosequences can be accomplished using a mathematical algorithm. Computerimplementations of these mathematical algorithms can be utilized forcomparison of sequences to determine sequence identity. Suchimplementations include, but are not limited to: CLUSTAL in the PC/Geneprogram (available from Intelligenetics, Mountain View, Calif.); theALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTAin the Wisconsin Genetics Software Package, Version 8 (available fromGenetics Computer Group (GCG), 575 Science Drive, Madison, Wis., USA).Alignments using these programs can be performed using the defaultparameters.

An agent that inhibits sonic hedgehog (SHH) activity (e.g., signaling)includes any agent (e.g., a peptide, protein, including antibodies,small molecule, drug, chemical, or nucleic acid, such as DNA or RNA)which inhibits the function or expression of sonic hedgehog (including,but not limited to, providing signal(s) in the patterning of the earlyembryo, such as patterning of the ventral neural tube, theanterior-posterior limb axis, and the ventral somites). Such agentsinclude, but are not limited to, an anti-sonic hedgehog antibody,cyclopamine (CPA), analogs thereof, such as cyclopamine-4-ene-3-one orother steroidal alkaloids. As used herein, “inhibit” refers to areduction (e.g., about 5%, about 10%, about 15%, about 20%, about 25%,about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,about 95% or about 100%) in the activity of sonic hedgehog as comparedto the activity of SHH in the absence of an agent that inhibits SHHactivity.

As described in Example 1 and 2 herein below, stem cells weredifferentiated into pancreatic progenitor cells and beta-cells in vitro.Briefly, stem cells were cultured in medium containing Activin-A (about0.5 ng/ml to about 200 ng/ml, such as about 50 ng/ml to about 100 ng/ml,including about 100 ng/ml) and Wnt3a (about 10 ng/ml to about 100 ng/ml,such as about 20 ng/ml to about 50 ng/ml or about 50 ng/mL) for about 3days, followed by about six days of culture in Activin A, Wnt 3a andanti-SHH antibody (about 2.5 mg/ml to about 10 ng/ml). The cellsobtained therefrom were next cultured in medium containing EGF (e.g.,about 5 to about 100 ng/mL, including about 50 ng/mL) for about 6 days.The cells obtained therefrom were then cultured in medium containingGDF-11 (e.g., about 5 to about 100 ng/mL, including about 50 ng/mL),exendin4 (e.g., about 5 nM to about 50 nM, including about 10 nM), andbetacellulin (e.g., about 10 ng/mL to about 100 ng/mL, including about50 ng/mL) for about six days.

Methods of identifying and subsequently separating differentiated cellsfrom their undifferentiated counterparts can be carried out by methodswell known in the art and described herein. Cells that have been inducedto differentiate can be identified by selectively culturing cells underconditions whereby differentiated cells outnumber undifferentiatedcells. Similarly, differentiated cells can be identified bymorphological changes and characteristics that are not present on theirundifferentiated counterparts, such as cell size, the number of cellularprocesses, the complexity of intracellular organelle distribution, andthe production of insulin or C-peptide and the secretion of insulin orC-peptide in response to glucose.

Also contemplated are methods of identifying differentiated cells bytheir expression of specific cell-surface markers such as cellularreceptors and transmembrane proteins. Monoclonal antibodies againstthese cell-surface markers can be used to identify differentiated cells.Detection of these cells can be achieved through fluorescence activatedcell sorting (FACS) and enzyme-linked immunosorbent assay (ELISA). Fromthe standpoint of transcriptional upregulation (or increase proteinexpression) of specific genes, differentiated cells often display levelsof gene expression that are different (increased or decreased expressionof mRNA or protein) from undifferentiated cells, such as insulin-1,insulin-2, glucagon, somatostatin, NeuroD1, Pdx-1, Ngn3, NRx6.1, NRx2.2,rfx-6, ptf1, glucokinase (glck), chromogranin, Maf, and/or glucosetransporter. Reverse-transcription polymerase chain reaction (RT-PCR)can be used to monitor such changes in gene expression duringdifferentiation. In addition, whole genome analysis using microarraytechnology can be used to identify differentiated cells.

Accordingly, once differentiated cells are identified, they can beseparated from their undifferentiated counterparts, if necessary. Themethods of identification detailed above also provide methods ofseparation, such as FACS, preferential cell culture methods, ELISA,magnetic beads, and combinations thereof.

Use of Stem/iPS Cell Derived Pancreatic Cells

The pancreatic progenitor or insulin producing cells of the inventioncan be used to repopulate a pancreas by either direct introduction intothe area of damage or by systemic administration, which allows the cellsto home to the area of damage. Accordingly, the invention providesmethods of treating a subject in need of pancreatic cells comprisingadministering to a subject an effective amount of the pancreaticprogenitor cells of the invention.

For the purposes described herein, either autologous, allogeneic orxenogeneic cells can be administered to a patient, either inundifferentiated, terminally differentiated or in a partiallydifferentiated form, genetically altered or unaltered, by directintroduction to a site of interest, e.g., on or around the surface of anacceptable matrix, or systemically, in combination with apharmaceutically acceptable carrier so as to repair, replace or promotethe growth of existing and/or new pancreatic cells.

Generally, the invention provides methods to treat a pancreaticdisorder. The term “pancreatic disorder” or “pancreatic disease” refersto a state where pancreatic function is impaired. Examples of“pancreatic disorders” or “pancreatic diseases” that can be treated withthe compositions and methods of the invention include, but are notlimited to, diabetes (including Type 1, Type 2, MODY and other geneticcauses of diabetes), obesity, pancreatic atresia, pancreas inflammation,alpha1-antitrypsin deficiency, acute, chronic or hereditarypancreatitis, pancreatic cancer (including endocrine tumors of thepancreas), pancreas malfunction due to cystic fibrosis or ShwachmanDiamond syndrome, pancreatic insufficiency or pancreatic enzymedeficiency, pancreatic cysts, hyperinsulinism, pancreatic digestivediseases, genetic disorders of the exocrine pancreas and pancreaticinjury, including, but not limited to, injury as a result of physicaltrauma (including, but not limited to, surgery), chemical, radiological,aging, and/or disease.

Administration of the Cells

Stem cell derived pancreatic progenitors can be administered to asubject by a variety of methods available to the art, including but notlimited to localized injection, catheter administration, systemicinjection, intraperitoneal injection, parenteral administration,intra-arterial injection, intravenous injection, transvascularinjection, intramuscular injection, subcutaneous placement/injection,surgical injection into a tissue of interest (e.g., injection into thepancreas) or via direct application to tissue surfaces (e.g., duringsurgery or on a wound).

Stem cell derived pancreatic progenitors can be administered eitherperipherally or locally through the circulatory system. “Homing” of stemcells would concentrate the implanted cells in an environment favorableto their growth and function. Pre-treatment of a patient withcytokine(s) to promote homing is another alternative contemplated in themethods of the present invention. Certain cytokines (e.g., cellularfactors that induce or enhance cellular movement, such as homing ofother stem cells, progenitor cells or differentiated cells) can enhancethe migration of iPS cell derived pancreatic progenitors or theirprogeny. Cytokines include, but are not limited to, stromal cell derivedfactor-1 (SDF-1), stem cell factor (SCF), angiopoietin-1,placenta-derived growth factor (PIGF) and granulocyte-colony stimulatingfactor (G-CSF). Cytokines also include any which promote the expressionof endothelial adhesion molecules, such as ICAMs, VCAMs and others,which facilitate the homing process.

Viability of newly forming tissues can be enhanced by angiogenesis.Factors promoting angiogenesis include, but are not limited to, VEGF,aFGF, angiogenin, angiotensin-1 and -2, betacellulin, bFGF, Factor X andXa, HB-EGF, PDGF, angiomodulin, angiotropin, angiopoetin-1,prostaglandin E1 and E2, steroids, heparin, 1-butyryl-glycerol andnicotinic amide.

Factors that decrease apoptosis can also promote the formation of newtissue, such as pancreatic tissues. Factors that decrease apoptosisinclude but are not limited to β-blockers, angiotensin-converting enzymeinhibitors (ACE inhibitors), AKT, HIF, carvedilol, angiotensin II type 1receptor antagonists, caspase inhibitors, cariporide and eniporide.

Exogenous factors (e.g., cytokines, differentiation factors (e.g.,cellular factors, such as growth factors or angiogenic factors thatinduce lineage commitment), angiogenesis factors and anti-apoptosisfactors) can be administered prior to, after or concomitantly with thecells.

A method to potentially increase cell survival is to incorporate cellsinto a biopolymer or synthetic polymer. Depending on the patient'scondition, the site of injection might prove inhospitable for cellseeding and growth because of scarring or other impediments. Examples ofbiopolymer include, but are not limited to, fibronectin, fibrin,fibrinogen, thrombin, collagen and proteoglycans. This can beconstructed with or without included cytokines, differentiation factors,angiogenesis factors or anti-apoptosis factors. Additionally, these canbe in suspension. Another alternative is a three-dimensional gel withcells entrapped within the interstices of the cell biopolymer admixture.Again cytokines, differentiation factors, angiogenesis factors,anti-apoptosis factors or a combination thereof can be included withinthe gel. These can be deployed by injection via various routes describedherein.

The cells can also be encapsulated with a capsule that is permeable tonutrients and oxygen while allowing appropriate cellular products (forexample, insulin in the case of islet cells) to be released into thebloodstream or to adjacent tissues. In one embodiment, the capsularmaterial is restrictive enough to exclude immune cells and antibodiesthat could reject and destroy the implant. Such encapsulation can beachieved using, for example, polymers (Chang, 2000). Such polymericencapsulation systems include, but are not limited to, alginate (e.g.,alginate bead), polysaccharide hydrogels, chitosan, calcium or bariumalginate, a layered matrix of alginate and polylysine, aphotopolymerizable poly(ethylene glycol) (PEG) polymer (Novocell, Inc.),a polyanionic material termed Biodritin (U.S. Pat. No. 6,281,341),polyacrylates, a photopolymerizable poly(ethylene glycol) polymer, andpolymers such as hydroxyethyl methacrylate methyl methacrylate. Anotherapproach to encapsulate cells involves the use of photolithographytechniques adapted from the semiconductor industry to encapsulate livingcells in silicon capsules that have pores only a few nanometers wide(Desai 2002). Also, suitable immune-compatible polycations, includingbut not limited to, poly-1-lysine (PLL) polycation or poly-1-ornithineor poly(methylene-co-guanidine) hydrochloride, may be used toencapsulate cells.

Additionally, cells can be encapsulated with biocompatible semipermeablemembranes to surround encapsulated cells, sometimes within a capillarydevice, to create a miniature artificial organ, such as one that wouldinclude functional pancreas or liver cells (e.g., a liver or pancreaticartificial device). This is often called macroencapsulation. Themembrane lets glucose, oxygen, and insulin pass in and out of the bloodstream, and preferably keeps out the antibodies and T cells of theimmune system, which may destroy the cells (e.g., islets). Suchmembranes can be used in a perfusion device, a capsule that is graftedto an artery where it makes direct contact with the body's circulatingblood; in this way, the device can draw nutrients from the blood andrelease insulin to circulate throughout the body. Another methodprovides for coating a small group of islet cells (macroencapsulation)or individual islet cells (microencapsulation) and implanting theminside the abdominal cavity. In these devices nutrients and insulinwould be exchanged by way of the body fluids permeating the tissues inwhich they are implanted.

The cells can also be administered in via a device or scaffoldingsubstance (that may or may not be a polymer) to contain the cells (e.g.,the cells can be placed in the device prior to implantation). In oneembodiment, this device/substance is retrievable. In another embodiment,it is absorbable. In another embodiment, a site may be createdsurgically to contain the cells. In one embodiment, the cells(pancreatic progenitor cells/insulin producing cells are transplantedwith additional cell types, including, but not limited to, mesenchymalstem cells or endothelial cells.

The quantity of cells to be administered will vary for the subject beingtreated. In a preferred embodiment, between about 10⁴ to about 10⁸, morepreferably about 10⁵ to about 10⁷ and most preferably, about 3×10⁷ stemcells and optionally, about 50 to about 500 μg/kg per day of a cytokinecan be administered to a human subject. However, the precisedetermination of what would be considered an effective dose may be basedon factors individual to each patient, including their size, age,disease or injury, amount of damage, amount of time since the damageoccurred and factors associated with the mode of delivery (directinjection—lower doses, intravenous—higher doses). Dosages can be readilyascertained by those skilled in the art from this disclosure and theknowledge in the art.

When administering a therapeutic composition of the present invention,it can generally be formulated in a unit dosage injectable form(solution, suspension, emulsion). The pharmaceutical formulationssuitable for injection include sterile aqueous solutions anddispersions. The carrier can be a solvent or dispersing mediumcontaining, for example, water, saline, phosphate buffered saline,polyol (for example, glycerol, propylene glycol, liquid polyethyleneglycol, and the like) and suitable mixtures thereof.

Additionally, various additives which enhance the stability, sterility,and isotonicity of the compositions, including antimicrobialpreservatives, antioxidants, chelating agents and buffers, can be added.Prevention of the action of microorganisms can be ensured by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, and the like. In many cases, it willbe desirable to include isotonic agents, for example, sugars, sodiumchloride, and the like. Prolonged absorption of the injectablepharmaceutical form can be brought about by the use of agents delayingabsorption, for example, aluminum monostearate and gelatin. According tothe present invention, however, any vehicle, diluent, or additive usedshould be compatible with the cells.

Sterile injectable solutions can be prepared by incorporating the cellsutilized in practicing the present invention in the required amount ofthe appropriate solvent with various amounts of the other ingredients,as desired.

In one embodiment, the cells described herein can be administeredinitially, and thereafter maintained by further administration of cells.For instance, the cells can be administered by one method of injection,and thereafter further administered by a different or the same type ofmethod.

Compositions are conveniently provided as liquid preparations, e.g.,isotonic aqueous solutions, suspensions, emulsions or viscouscompositions, which may be buffered to a selected pH. Liquidpreparations are normally easier to prepare than gels, other viscouscompositions and solid compositions. Additionally, liquid compositionsare somewhat more convenient to administer, especially by injection.Viscous compositions, on the other hand, can be formulated within theappropriate viscosity range to provide longer contact periods withspecific tissues.

The choice of suitable carriers and other additives will depend on theexact route of administration and the nature of the particular dosageform, e.g., liquid dosage form (e.g., whether the composition is to beformulated into a solution, a suspension, gel or another liquid form,such as a time release form or liquid-filled form).

Solutions, suspensions and gels normally contain a major amount of water(preferably purified, sterilized water) in addition to the cells. Minoramounts of other ingredients such as pH adjusters (e.g., a base such asNaOH), emulsifiers or dispersing agents, buffering agents,preservatives, wetting agents and jelling agents (e.g.,methylcellulose), may also be present. The compositions can be isotonic,i.e., they can have the same osmotic pressure as blood and lacrimalfluid.

The desired isotonicity of the compositions of this invention may beaccomplished using sodium chloride, or other pharmaceutically acceptableagents such as dextrose, boric acid, sodium tartrate, propylene glycolor other inorganic or organic solutes. Sodium chloride is preferredparticularly for buffers containing sodium ions.

Viscosity of the compositions, if desired, can be maintained at theselected level using a pharmaceutically acceptable thickening agent.Methylcellulose is preferred because it is readily and economicallyavailable and is easy to work with. Other suitable thickening agentsinclude, for example, xanthan gum, carboxymethyl cellulose,hydroxypropyl cellulose, carbomer, and the like. The preferredconcentration of the thickener will depend upon the agent selected andthe desired viscosity. Viscous compositions are normally prepared fromsolutions by the addition of such thickening agents.

A pharmaceutically acceptable preservative or cell stabilizer can beemployed to increase the life of the compositions. Preferably, ifpreservatives are necessary, it is well within the purview of theskilled artisan to select compositions that will not affect theviability or efficacy of the cells as described in the presentinvention.

Those skilled in the art will recognize that the components of thecompositions should be selected to be chemically inert. This willpresent no problem to those skilled in chemical and pharmaceuticalprinciples, or problems can be readily avoided by reference to standardtexts or simple experiments (not involving undue experimentation), fromthis disclosure and the documents cited herein.

Monitoring of Subject after Administration of Cells

Following transplantation, the growth or differentiation of theadministered cells or or the therapeutic effect of the cells may bemonitored. For example, blood glucose, serum glucose, HbA1c (a measureof glycosylated protein) and/or serum insulin may be monitored.

Following administration, the immunological tolerance of the subject tothe cells may be tested by various methods known in the art to assessthe subject's immunological tolerance to the cells. In cases where thesubject's tolerance of the cells is suboptimal (e.g., the subject'simmune system is rejecting the exogenous cells), therapeutic adjunctimmunosuppressive treatment, which is known in the art, of the subjectmay be performed.

EXAMPLES

The following examples are provided in order to demonstrate and furtherillustrate certain embodiments and aspects of the present invention andare not to be construed as limiting the scope thereof.

Example 1 Differentiation of Stem Cells to Pancreatic Cells Materialsand Methods

Lentivirus production: Each recombinant lentivirus expressing humanOct4, Sox2, Nanog, and Lin28 was generated by transfecting theconstructors (Addgene, 16579, 16577, 16578, 16580) together withpackaging plasmid pΔNRF and MD.G into 293FT cells. Briefly, 4.5×10⁶293FT cells were seeded using DMEM medium supplemented with 10% FBS in15 cm plates. The transfection was conducted the next day using acalcium-phosphate-mediated method. Twenty hours after transfection, themedium was changed to DMEM with 2% FBS. Viral supernatants wereharvested at 24 and 48 hours post transfection and concentrated byultracentrifugation at 22,000 rpm for 2 hours.

Derivation of iPS cell lines: 1.25×10⁵ neonatal human dermal fibroblast(NHDF, Lonza) cells were seeded on a gelatin coated 6-well plate the daybefore lentiviral transduction in growth medium containing high-glucoseDMEM, 10% FBS, 1×NEAA. A mixture of 4 different recombinant lentivirusesexpressing human Oct4, Sox2, Nanog, and Lin28 was used to infect theNHDF cells in the presence of 8 μg/ml polybrene (Sigma). After overnightincubation with the mixture, the medium containing viruses was replacedwith fresh growth medium. Four days after transduction, 5.5×10⁴ cellswere collected by trypsin (Invitrogen) digestion and transferred ontoirradiated mouse embryonic fibroblast (MEF) cells in each well of 6-wellplates with human embryonic stem (hES) cell medium (DMEM/F12 mediumcontaining 20% knockout serum replacement, 0.1 mM nonessential aminoacids, L-glutamine, 0.1 mM β-mercaptoethanol and 4 ng/ml basicfibroblast growth factor). Colonies with typical hES cell morphologyappeared 15 days post-transduction and were picked for expansion on day26.

Cell culture and differentiation: iPS cell lines were cultured in iPSmedium (DMEM/F12 medium containing 20% knockout serum replacement, 0.1mM nonessential amino acids, L-glutamine, 0.1 mM β-mercaptoethanol and100 ng/ml basic fibroblast growth factor). Cultures were maintained onirradiated primary (MEF) feeders and passaged enzymatically using 10μg/ml collagenase IV (Invitrogen).

Differentiation was achieved using embryoid body (EB) formation.Spontaneous differentiation was initiated by dissociating human iPScells using collagenase, and putting them into 24-well Ultra-lowattachment plate in differentiation medium (DMEM supplemented with 20%FBS, 1 mM L-glutamine, 0.1 mM nonessential amino acids, 0.1 mMβ-mercaptoethanol). Directed differentiation was conducted in 2% FBSdifferentiation medium with the following steps: Stage 1: from day 0 today 3, the culture was supplemented with 100 ng/ml Activin A and 50ng/ml Wnt3a. Stage 2: From day 3 to day 9, the medium was supplemented2.5 mg/ml anti-human Shh in addition to Activin A and Wnt3a. Stage 3:from day 9 to day 15, the medium was supplemented with 50 ng/ml hEGF and50 ng/ml heparin sulfate. Stage 4: from day 15 to day 21, the mediumcontained 50 ng/ml hGDF-11, 50 ng/ml hBetacellulin and 10 nM Exendin.All cytokines except Exendin (Sigma) were from R&D Systems. Thestimulation schedule is as shown in Table 1. The medium was refreshedevery 3 days. Samples of EBs were harvested every 3 days for analysis ateach stage to identify specific cell populations.

RNA Extraction and gene expression analysis: Total RNA was extractedfrom cell samples using the RNeasy Micro kit (Qiagen) according to theprotocol provided in the kit and 0.5-5 μg of the extracted total RNA wasused in reverse transcription to synthesize cDNA using the SuperScriptIII First-stand Synthesis System for RT-PCR kit (Invitrogen).Quantitative PCR (qPCR) was carried out with cDNA using SYBR Green(Applied Biosystems) on an Eppendorf Mastercycler (realplex²).

Immunofluorescence: For iPS cell line characterization, colonies growingon feeders were fixed using 4% paraformaldehyde and then washed withPBS. Immunostaining was performed using primary antibodies against Oct4(Chemicon), Nanog (R&D systems), Sox2 (R&D systems), Tra-1-81 (Chemicon)for overnight at 4° C. first, followed by anti-mouse or anti-goat AlexaFluor 488 for 1 hour in the dark after washing with PBS plus 0.05%Tween-20. For SSEA-4 staining, after fix, PE conjugated antibody wasperformed directly for 1 hour in the dark after washing with PBS plus0.05% Tween-20.

For differentiation analysis, harvested EBs were washed three times inDulbecco's phosphate buffered saline (PBS), fixed for 30 minutes in 4%paraformaldehyde, and processed for paraffin embedding. Sections (7 μm)were retrieved with Antigen Retrieval Reagent (R&D systems) at 95° C.for 5 minutes, blocked with PBS plus 1% BSA, 10% donkey serum and TritonX-100 for 30 minutes, and incubated with primary antibody againstproinsulin (rat anti-human proinsulin, GN-ID4, DSHB) and Pdx-1(Biotinylated goat anti-human Pdx-1) (R&D) for 2 hour at roomtemperature, followed by streptavidin Alexa Fluor 488 for Pdx-1 andsecondary antibodies anti-rat IgG-PE (BD Bioscience) for proinsulin.Staining was observed by fluorescence microscopy.

Detection of C-peptide concentration in culture supernatants by ELISA:Culture supernatants were harvested at different time points and storedat −80° C. until use. C-peptide concentration was detected according tothe instructions from the human C-peptide kit (Millipore).

Transplantation of differentiated L-1 cells into mice: Mice weresocially housed and participated in a complete enrichment program.Dietary enrichment included provision of black oil sunflower seeds(Bio-Serv, Frenchtown, N.J.) and Enrich Mix (1922 Harlan Teklad,Madison, Wis.) daily. Environmental enrichment included a crawl ball,polycarbonate igloo, or paper hut.

Diabetes Induction in Nude Mice: Adult male nude mice, 29.7-33.5 grams,were obtained from the Charles River Laboratories (Wilmington, Mass.,USA). Diabetes induction was accomplished using a single IP infusion ofstreptozotocin (STZ, Zanosar; Sicor Pharmaceuticals, Irvine, Calif.,USA) 240 mg/kg bolus. Following the administration of STZ, animals wereclosely monitored for adverse events Animals received supportivehydration (1-3 ml normal saline IP) concomitantly and as clinicallyindicated post STZ injection. Blood glucose and weight were measureddaily or as clinically indicated from STZ to the scheduled experimentalendpoint. Blood glucose levels were measured by bleeding the tail vein.Mice with a blood glucose level >300 mg/dL for 2 consecutive days wereconsidered diabetic, at which time insulin injections were initiated. Indiabetic mice, 0.5U glargine (Lantus, Aventis, Parsippany, N.J.) wasinjected subcutaneously, daily or as clinically indicated, until betacell transplant, or in some experiments until glucose levels werestabilized below 350 mg/dL. Blood collection, via tail or facial veinbleed, was performed approximately every 14 days for RIA analysis forhuman C-peptide using the Millipore human RIA kit according to themanufacturers instructions.

Transplant Under Renal Capsule of Nude Mice: Isoflurane was deliveredvia precision anesthetic vaporizer for anesthesia. After full asepticpreparation using Technicare surgical scrub (C are Tech Laboratories, StLouis, Mo.), the mouse was placed laterally on surgical field. Using asterile dissecting forceps and scissors a 1 cm incision in the skin andperitoneum was created to expose the kidney. The kidney was gentlyexternalized using palpation. A small nick (1-3 mm) to the kidneycapsule was made and the collected differentiated iPS cells,approximately 1-2×10⁶ cells (FIG. 3) or 3-8×10⁶ (FIG. 4) cells wereplaced under the kidney capsule, using PE160 tubing attached to aHamilton syringe. The kidney was reintroduced into the peritoneum, themuscle layer was approximated, and the skin layer was closed withabsorbable suture. Analgesia was accomplished using ketoprofen 5 mg/kgSC a single dose pre-operatively, and as needed post operatively.

Transplant in Epididymal Fat Pad of Nude Mice: Isoflurane was deliveredvia precision anesthetic vaporizer for anesthesia. After full asepticpreparation using Technicare surgical scrub (C are Tech Laboratories, StLouis, Mo.), the mouse was placed dorsally on surgical field. Using asterile dissecting forceps and scissors a 1 cm incision in the skin andfascia was created on the ventral midline in the groin area. Theepididymal fad pad (EFP) was gently exposed and kept moist withphysiologic saline. A purse string suture was used at the periphery ofthe EFP to create a pouch. Between 4 and 8×10⁶ differentiated iPS cellswere loaded into PE160 tubing, the PE tubing placed into the pouch, andcells were delivered to the EFP pouch. The purse string was ligated,closing the opening and marking site of implantation. The fascia andskin were closed with absorbable suture. Analgesia was accomplishedusing ketoprofen 5 mg/kg SC a single dose pre-operatively, and as neededpost operatively.

Nephrectomy: Isoflurane was delivered via precision anesthetic vaporizerfor anesthesia. After full aseptic preparation using Technicare surgicalscrub (C are Tech Laboratories, St Louis, Mo.), the mouse was placedlaterally on surgical field. Using a sterile dissecting forceps andscissors a 1 cm incision was made through skin and muscle at theprevious incision site. The kidney was gently exposed and the renalvessels were ligated. The kidney was then removed, the muscle layer wasapproximated and the skin layer was closed with absorbable suture.Analgesia was accomplished using ketoprofen 5 mg/kg SC a single dosepre-operatively, and as needed post operatively. The graft and kidneywere collected for immunohistochemistry and PCR analysis.

An efficient method for differentiating human embryonic stem (hES) cellsinto pancreatic endoderm cells based on sequential exposure to cytokinesthat regulate mammalian pancreatic fate in vivo was developed usingfewer cytokines than other published protocols (Table 1).

TABLE 1 Strategy for differentiation of pluripotent cells intoinsulin-producing cells. Cytokine Fate Induced Stage 1 Day 0-3 100 ng/mlActivin A Endoderm 50 ng/ml Wnt 3a Stage 2 Day 3-9 100 ng/ml Activin ADefinitive 50 ng/ml Wnt 3a endoderm 2.5 ug/ml anti-Shh Pancreaticendoderm Stage 3 Day 9-15 50 ng/ml EGF Expansion of Pancreatic endodermStage 4 Day 15-21 50 ng/ml GDF-11 Pancreatic cells 50 ng/ml Maturationof β Betacellulin cells 10 nM Exendin4This four step method was used to differentiate the hES cell line H9into insulin producing pancreatic cells. Specific differentiatedintermediates were identified by sequential expression of a series oftranscriptional factors. Foxa2 was used as a marker for definitiveendoderm. Following maturation to pancreatic endoderm, expression of thetranscription factors Pdx-1 and Ptf1a were observed, while mature betacells were indicated by the expression of Pdx-land insulin.Differentiating H9 cells exhibited a similar sequential gene expressionpattern to the HSF-6 hES line under the same conditions (not shown) andduring in vivo mouse development (Habener et al. (2005) and Jensen(2004) (see FIG. 6)). These data suggest that pancreatic development isoccurring in embryoid bodies (EBs), and that the differentiation schemeis robust enough to direct differentiation in multiple pluripotentlines.

The four step protocol was used to direct differentiation of L-1 iPScells. The L-1 cell line, derived by lentiviral infection of NHDFs, iskaryotypically normal (data not shown), and has similar morphology andpluripotency marker gene expression to hES cells as shown byimmunostaining (FIG. 7 a). Two cell lines derived from NHDFs showedsimilar pluripotent marker gene expression to H9 hES cells (FIG. 7 b).NHDF-derived lines (L-1 and L-2) and H9 cells express Oct4, Nanog andLin28, as well as hTert, and several other pluripotency-associated genesin contrast to the expression of only hcMyc and the control marker GAPDHobserved in the parent NHDF cells.

To demonstrate the pluripotency of the L-1 iPS line in vitro,unstimulated differentiation to representative cell types of the threegerm cell layers was assessed (FIG. 8). The differentiation protocoldeveloped for hES cells was applied to the L-1 cells. Embryoid Bodycultures were established for 21 days with sequential cytokinestimulation (see Table 1). The L-1 cells were shown to differentiateinto beta-like cells with the capacity for insulin secretion aftermaturation through the differentiation steps previously observed duringculture of H-9 cells, consistent with normal pancreatic development(FIG. 2 a). qPCR analysis for stage-specific markers showed sequentialdifferentiation of the L-1 cells into endoderm, definitive endoderm, andthen to pancreatic endoderm. High levels of the endodermal markers,CXCR4 and ECadherin, were detected within the first week. Immediatelythereafter, definitive endodermal markers, Foxa2 and Sox17 wereexpressed at peak levels. Finally, the pancreatic endodermal markersPdx-1 and Ptf1a were detected in the last week of differentiation.Markers related to mature beta cells and insulin secretion peaked in asimilar time frame (FIG. 2 b). Since pancreatic precursors transientlyexpress Pdx-1 in vivo, followed by re-expression in matureinsulin-producing beta cells, the expression of Pdx-1 and insulin wasexamined by immunostaining of cross-sections of EBs at day 21 (FIG. 2c). Examination of the cross-sections shows that no single cellexpressed both of these markers, suggesting that precursors and immatureinsulin-producing cells, but not mature beta cells were present in thesecultures. However, differentiated L-1 cells were actively secretinginsulin, as measured by ELISA assay to detect human C-peptideconcentration in the supernatant at different time points. The level ofC-peptide was below the threshold of detection in undifferentiated day 0L-1 cells. C-peptide secretion was detected in the medium of later stagecells beginning at day 15 of culture, suggesting that beta-like cellswere present.

While a few groups have been successful in generating iPS-derived cellswith evidence of insulin production in vitro, the function of these celllines has not been tested in vivo. Differentiated L-1 cells generated inthe studies above were transplanted under the kidney capsule ofstreptozotocin (STZ) induced diabetic mice to test the ability of thebeta-like cells to reduce hyperglycemia and reverse the diabetic state.Initially, cells from different time points were transplanted todetermine the optimal timing and cell doses. In this initial phase,insulin injections were withdrawn at the time of transplantation. Bloodglucose levels and body weights of STZ-treated mice were measured dailyover a three week period after transplantation (FIGS. 3 a and 3 b).Cells transplanted at day 15 of EB culture showed transient reduction ofglucose levels to normoglycemia, and levels over the three weeks, thoughelevated, were maintained similar to those with daily insulin injectionsprior to transplantation. No exogenous insulin was given after graftexplant, body weights decreased, suggesting the differentiated L-1 graftcontributed to glycemic control and metabolic stability prior toexplant.

In the second phase of the study, mice were maintained aftertransplantation with daily insulin administration until glucose wasmeasured to be lower than 350 mg/dL, a level consistent with thesurvival of the mouse. Cultures of differentiated cells were grown instirred bioreactors to facilitate transplantation of larger cell doses,and in larger numbers of mice, using the same differentiation scheme.Differentiated L-1 cells (4-8×10⁶) were transplanted either under thekidney capsule, or into the epidermal fat pad (EFP). Blood glucoselevels and body weights were measured daily (FIGS. 4 a and 4 b). In thisexperiment, transplantation of differentiated iPS cells reverseddiabetes to below the target level of 350 mg/dL in five of six micetested, yielding stable glucose levels for more than 3 weeks, althoughthe levels varied among the mice. Human C-peptide levels were measuredin a human specific radioimmunoassay (RIA) to ascertain whether insulinsecretion was human graft-dependent or produced by the murine host.Human C-peptide levels were measured in a human specificradioimmunoassay (RIA) to ascertain whether insulin secretion was humangraft-dependent or produced by the murine host. Human C-peptide wasdetected in at least one of the six mice tested, supporting theconclusion that insulin production from the graft was responsible forreduction of glucose levels and maintenance of body weight. The graftwas then removed and immunostaining for pdx-1 and proinsulin wasperformed. FIG. 5 shows that there was co-expression of both of thesemarkers in cells from transplanted grafts supporting the conclusion thatmature beta cells formed in vivo. This correlated with normalization ofglucose in this mouse. No insulin-positive cells were detected in thegraft from the mouse with the highest glucose levels, although qPCRanalysis demonstrated insulin expression in the grafts.

These studies establish the feasibility of differentiating iPS cellsderived from human dermal fibroblasts to insulin-producing cells invitro using a novel four-step protocol. The beta-like cells secretebiologically relevant levels of insulin when transplanted in STZ-treateddiabetic mice, demonstrated by their capacity to reverse diabetes. Theobservations, in conjunction with ES cell and in vitro iPS studies ofothers (Shim (2007); Jiang et al (2007); Philips et al. (2007); D'Amouret al. (2005); D'Amour et al. (2006); Kroon et al. (2008); Zhang et al.(2009); Tateishi et al. (2008); Maehra et al. (2009)), support thehypothesis that differentiation of pluripotent cells in vitro faithfullyrecapitulates in vivo pancreatic developmental patterns (Habener et al.(2005) and Jensen (2004)). Insulin secretion was observed at laterstages of differentiation, but glucose-stimulated insulin secretion wasnot observed either in the cultures analyzed in this study, or instudies with ES cells. The glucose insensitivity of insulin-producingcells in late stage iPS cultures is similar to the phenotype of fetalpancreatic islet cells as the progression to glucose-responsive betacells occurs after birth. Additional evidence that precursors, but notbeta cells were present in EBs prior to transplantation comes from thelack of expression of pdx-1 in insulin producing cells.Immunohistochemical data showed that insulin-producing cells in EBs atthe time of transplantation did not co-express pdx-1, as would beexpected in mature beta cells. These observations support theinterpretation that mature beta cells were not present at day 15 ofdifferentiation even though C-peptide was present in the supernatant ofthese cultures. However, since insulin/pdx-1 co-expressing cells werepresent in the grafts after excision, further maturation could aftertransplantation. The transplantation of mature beta cells is notrequired to achieve glucose regulation by less mature cells in vivo,since glucose levels were significantly reduced in engrafted mice asearly as day 12 after transplant.

Methods of differentiation previously used to generate insulin-producingcells have been complicated, requiring multiple steps and the additionof many individually added cytokines, without significant expansion ofcell numbers (D'Amour et al. (2006)). Simplified differentiation schemessuch as the methods used here have significant advantages, requiringfewer individual recombinant factors which will facilitatestandardization and reduce costs of the scale-up of cultured cells fortransplantation. Further, the culture of cell clusters like EBs insuspension is amenable to larger scale bioreactors. In all of theprevious studies, inhibition of Shh signaling from definitive endoderm,a step required for the specification of pancreas, was achieved by theaddition of cyclopamine, a toxic chemical. In this study, similarresults were achieved by the addition of an inhibiting antibody thatblocks Shh signaling without associated toxicity.

In summary, it is reported herein for the first time, a robust andscalable iPS differentiation method to generate insulin-producing cellsthat rescue diabetes. This study demonstrates that patient-specificcells may be useful both as a model of human pancreatic development forinvestigating the events leading to type 1 diabetes, and for thegeneration of therapies that will provide sufficient islet replacementcells for the treatment of type 1 diabetes.

Islet replacement therapy for type 1 diabetes is a promising approachfor restoring insulin-producing beta cells; however, broadimplementation of this strategy is currently not feasible due to thescarcity of donor tissues. A potential solution to this problem would bethe directed differentiation of human pluripotent stem cells, includingembryonic stem (ES) cells and induced pluripotent stem (iPS) cells intoinsulin-producing cells. While others have succeeded in deriving cellsthat secrete insulin, protocols have been complex and therefore notoptimal for clinical translation. A streamlined, broadly applicablefour-step differentiation process has been developed demonstrating thathES cells and human iPS cells from dermal fibroblast cells generateinsulin-producing cells. Further, the biological functionality of thesedifferentiated iPS cells was validated by transplantation into diabeticmice and subsequent reversal of diabetes in vivo. Thus, the studyprovides the first direct in vivo evidence that insulin-producing cellscan be generated from iPS cells using a robust process.

Example II Bioreactor Cultured Human iPS Cell Derived PancreaticProgenitors Enhance Diabetic Mice Survival Materials and Methods

Cell culture: iPS cell lines were cultured in iPS medium (DMEM/F12medium containing 20% knockout serum replacement, 0.1 mM nonessentialamino acids, L-glutamine, 0.1 mM β-mercaptoethanol and 100 ng/ml basicfibroblast growth factor). Cultures were maintained on irradiatedprimary (MEF) feeders and passaged enzymatically using 10 μg/mlcollagenase IV (Invitrogen).

Directed differentiation: Directed differentiation was achieved usingembryoid body (EB) formation (Embryoid Body (EB) culture is used toexamine the differentiation potential of the embryonic stem (ES) cellline. The cells are grown using low-attachment dishes in the presence ofcomplete growth medium. This process induces differentiation, permitssuspension culture, and causes the cells to form aggregates). Thedifferentiation was initiated by harvesting human iPS colonies usingcollagenase and shearing with a serological pipet. Differentiation wascarried out in suspension culture of EBs using a 4-step process indifferentiation medium (DMEM supplemented with 2% FBS, 1 mM L-glutamine,0.1 mM nonessential amino acids, 0.1 mM β-mercaptoethanol) with thefollowing steps: Stage 1: from day 0 to day 3, the culture wassupplemented with 100 ng/ml Activin A and 50 ng/ml Wnt3a. Stage 2: Fromday 3 to day 9, the medium was supplemented 2.5 mg/ml anti-human Shh inaddition to Activin A and Wnt3a. Stage 3: from day 9 to day 15, themedium was supplemented with 50 ng/ml hEGF and 50 ng/ml heparin sulfate.Stage 4: from day 15 to day 21, the medium contained 50 ng/ml hGDF-11,50 ng/ml hBetacellulin and 10 nM Exendin. All cytokines except Exendin(Sigma) were from R&D Systems. The stimulation protocol is as shown inFIG. 1. The medium was replaced every 3 days. Samples of EBs wereharvested every 3 days for analysis to identify specific cellpopulations, and for cell counts and viability assays. Differentiationwas conducted in the following suspension cultures: 1) 24-well Ultra-lowattachment plate or 10 cm Petri dishes (Corning) with 1 or 10 ml mediumfor static culture; 2) 100 ml stirred bioreactor (Wheaton Science) largescale culture with 70 rpm rotation by a low speed stirrer (WheatonScience). All incubations were carried out in a 5% CO₂ incubator at 37°C.

Biochemical Assays: EB samples were taken from the suspension culturesat the different time points and were extensively washed with PBS 3times. Cells were then lysed in different buffers from DNA (DNAeasyblood & tissue kit, QIAGEN) and protein assay kits and follow themanufacturer's instructions for the assays (BCA protein assay kit,thermo Scientific).

EB number, size, cell density and viability: Samples from all culturesystems were collected, EB sizes from different culture systems werecompared using inverted light microscopy (ZEISS, Axiovert 200M). Tocount cells, samples of EBs were treated with Accumax (Sigma) at 37° C.for 30 minutes and counted using Trypan blue dye exclusion on ahemacytometer.

RNA Extraction and gene expression analysis: Total RNA was extractedfrom cell samples using the RNeasy Micro kit (Qiagen) according to theprotocol provided in the kit and 0.5-5 μg of the extracted total RNA wasused in reverse transcription to synthesize cDNA using the SuperScriptIII First-stand Synthesis System for RT-PCR kit (Invitrogen).Quantitative PCR (qPCR) was carried out with cDNA using SYBR Green(Applied Biosystems) on an Eppendorf Mastercycler (realplex2). Primersused for amplification are listed in Table 2.

TABLE 2 Primer sets Gene marker Sets sequence GAPDH FGAGTCAACGGATTTGGTCGT (SEQ ID NO: 1) RGACAAGCTTCCCGTTCTCAG (SEQ ID NO: 2) Sox17 FCGCACGGAATTTGAACAGTA (SEQ ID NO: 3) RGGATCAGGGACCTGTCACAC (SEQ ID NO: 4) Foxa2 FATTGCTGGTCGTTTGTTGTG (SEQ ID NO: 5) RTACGTGTTCATGCCGTTCAT (SEQ ID NO: 6) Pdx-1 F CATTGGAAGGCTCCCTAACACA(SEQ ID NO: 7) R GGCATCAATTTCACGGGATC (SEQ ID NO: 8) Insulin  FCTACCTAGTGTGCGGGGAAC (SEQ ID NO: 9) RGCTGGTAGAGGGAGCAGATG (SEQ ID NO: 10)

Transplantation of differentiated L-1 cells into mice: Mice weresocially housed and participated in a complete enrichment program.Dietary enrichment included provision of black oil sunflower seeds(Bio-Serv, Frenchtown, N.J.) and Enrich Mix (1922 Harlan Teklad,Madison, Wis.) daily. Environmental enrichment included a crawl ball,polycarbonate igloo, or paper hut.

Diabetes Induction in Nude Mice: Adult male nude mice, 29.7-33.5 grams,were obtained from the Charles River Laboratories (Wilmington, Mass.,USA). Diabetes induction was accomplished using a single IP infusion ofstreptozotocin (STZ, Zanosar; Sicor Pharmaceuticals, Irvine, Calif.,USA) 240 mg/kg bolus. Following the administration of STZ, animals wereclosely monitored for adverse events Animals received supportivehydration (1-3 ml normal saline IP) concomitantly and as clinicallyindicated post STZ injection. Blood glucose and weight were measureddaily or as clinically indicated from STZ to the scheduled experimentalendpoint. Blood glucose levels were measured by bleeding the tail vein.Mice with a blood glucose level >300 mg/dL for 2 consecutive days wereconsidered diabetic, at which time insulin injections were initiated. Indiabetic mice, 0.5U glargine (Lantus, Aventis, Parsippany, N.J.) wasinjected subcutaneously, daily or as clinically indicated, until betacell transplant, or in some experiments until glucose levels werestabilized below 350 mg/dL. Blood collection, via tail or facial veinbleed, was performed approximately every 14 days for RIA analysis forhuman C-peptide using the Millipore human RIA kit according to themanufacturers instructions.

Transplant Under Renal Capsule of Nude Mice: Isoflurane was deliveredvia precision anesthetic vaporizer for anesthesia. After full asepticpreparation using Technicare surgical scrub (C are Tech Laboratories, StLouis, Mo.), the mouse was placed laterally on surgical field. Using asterile dissecting forceps and scissors a 1 cm incision in the skin andperitoneum was created to expose the kidney. The kidney was gentlyexternalized using palpation. A small nick (1-3 mm) to the kidneycapsule was made and the collected differentiated iPS cells,approximately 1-2×10⁶ cells or 3.5-5.5×10⁶ cells were placed under thekidney capsule, using PE160 tubing attached to a Hamilton syringe. Thekidney was reintroduced into the peritoneum, the muscle layer wasapproximated, and the skin layer was closed with absorbable suture.Analgesia was accomplished using ketoprofen 5 mg/kg SC a single dosepre-operatively, and as needed post operatively.

Transplant in Epididymal Fat Pad of Nude Mice: Isoflurane was deliveredvia precision anesthetic vaporizer for anesthesia. After full asepticpreparation using Technicare surgical scrub (C are Tech Laboratories, StLouis, Mo.), the mouse was placed dorsally on surgical field. Using asterile dissecting forceps and scissors a 1 cm incision in the skin andfascia was created on the ventral midline in the groin area. Theepididymal fad pad (EFP) was gently exposed and kept moist withphysiologic saline. A purse string suture was used at the periphery ofthe EFP to create a pouch. Between 4 and 8×10⁶ differentiated iPS cellswere loaded into PE160 tubing, the PE tubing placed into the pouch, andcells were delivered to the EFP pouch. The purse string was ligated,closing the opening and marking site of implantation. The fascia andskin were closed with absorbable suture. Analgesia was accomplishedusing ketoprofen 5 mg/kg SC a single dose pre-operatively, and as neededpost operatively.

Results

The Growth of Differentiated iPS Cell in Stirred Bioreactor Culture isComparable with Static Culture and Provides for Large Scale Culture.

Differentiation of hESCs can be initiated by the formation of embryoidbodies (EBs) in suspension. Undifferentiated L-1 cells were removed fromthe maintenance culture by collagenase and shearing to clusters ofapproximately 100 cells. Directed differentiation was achieved throughsuspension culture in two different systems, 10 mL ultralow-attachmentdishes (Corning) for static suspension culture and 100 mL bioreactorchambers (Wheaton) for stirred suspension cultures. The cells wereinoculated at same density (5.3×10⁵ cells/ml) on day 0 in parallelcultures. Samples of EBs were collected at several time points tocompare growth and differentiation parameters. The observation of EBmorphology by phase contrast microscopy showed fewer aggregates of EBsin the bioreactor cultures than static cultures (FIG. 9 a). Comparablecell numbers were obtained at the later stages of differentiation andsimilar viability levels were observed in the two differentiationconditions (FIG. 9 b), which indicates that the EBs formed from iPScells under 70 rpm stiffing in the bioreactor were not harmed byincreased volume or shear forces from the apparatus. However, the datashow that EB numbers decreased significantly after transferring the iPScolonies from maintenance culture (day 0) to directed differentiation(day 3) both in static and bioreactor cultures (FIG. 9 c), possibly dueto aggregation of individual EBs. Despite the similarity in EB numbers,aggregation of EBs in static culture appears to increase the average EBsize over the bioreactor culture, especially midway throughdifferentiation (day 9 in FIG. 9 a) in this study. The observeddifference in EB diameter between the static and bioreactor cultures wasconsistent with the observation of EB morphology by microscopy (FIGS. 9d, 9 e and 9 a). However, statistical analysis did not reveal asignificant difference between the average EB size under the two cultureconditions over the full differentiation course (p=0.0508). Thus, littleor no aggregation was seen in the bioreactor cultures, and the EBs instatic culture required manual dissociation when changing medium toprotect the culture from forming too large aggregates to support cellsurvival. Total DNA and protein was measured in samples taken at eachtime point (FIGS. 9 f, 9 g) indicating higher concentrations in staticculture than bioreactor during the early stages of differentiation (fromday 0 to day 15), and similar concentrations during later stages (fromday 15 to day 21), which is consistent with the cell number detection.These observations showed iPS cell differentiation cultures could beamplified simply from small static conditions to stirred large-scalebioreactors, which can provide large scale differentiated cells forclinic transplantation. Homogenous nutrient and gas perfusion inbioreactors can provide better conditions at later stages ofdifferentiation. Stirred bioreactors provide uniform nutrient supply,including added cytokines, O₂ and CO₂ perfusion, and pH levels incultured cells.

Bioreactor Culture Enhanced iPS Cell Differentiation into PancreaticProgenitor Cells.

Bioreactor culture of iPS cell differentiation demonstrated comparableEB growth when compared with static cultures. In order to addresswhether the differentiation to pancreatic cells in the large-scalebioreactor culture was efficient, differentiation was measured byexpression of specific pancreatic developmental markers, includingdefinitive endoderm markers Foxa2 and Sox17; as well as pancreaticendoderm marker Pdx-1. Compared with the static culture, the results forbioreactor differentiation demonstrated the same pattern of developmentof pancreatic progenitor cells, which mimics in vivo pancreaticdevelopment (FIG. 10). Bioreactor cultures expressed higher levels ofPdx-1, which represents the differentiation of pancreatic progenitorcells. Published reports indicate that the transplantation of pancreaticprogenitor cells reversed diabetes in mice efficiently. Since thedifferentiation of iPS cells in stirred bioreactor culture generatedlarge numbers of cells with characteristics of pancreatic progenitors(FIG. 10 b), the transplantation of iPS-derived cells from bothconditions into diabetic mice were compared to determine their abilityto regulate glucose in vivo.

Bioreactor Cultured iPS Derived Cells Enhanced Diabetic Mice Survival inVivo.

In a transplantation study, the timing of differentiation of iPS cellswas optimized for transplantation into mice made diabetic by theinjection of the beta cell toxin streptozoticin (STZ) using small-scalestatic cultures. Blood glucose levels and body weights were measureddaily over a three week period without insulin administration aftertransplantation. Mice transplanted with day 15 static cultured cellsshowed a transient decrease in blood glucose levels. This suggested thatthe transplanted graft helped regulate glucose, because the levelsobserved were consistent with diabetic mice receiving insulinadministration. Therefore, in the second phase of transplantation study,day 15 iPS-derived cells from both 10 ml static culture and 100 mlstirred bioreactor were transplanted to diabetic mice. Mice transplantedwith cells from static cultures reduced blood glucose to levels thatsupported maintenance of body weight and survival (FIG. 11 a and datanot shown). In contrast, mice transplanted with cells from stirredbioreactors reduced blood glucose levels to normoglycemia in five of sixmice tested (FIG. 11 b).

Furthermore, in the same study, inoculated cell densities were comparedfor static and stirred cultured, to determine whether stirredbioreactors supported both higher cell volumes as well as higher densitywithout loss of function in vivo. Three groups of differentiationcultures were set up: group 1, 10 ml static culture (at high density ofabout 1.4×10⁶/ml); group 2, 100 ml stirred bioreactor culture at highdensity (about 1.4×10⁶/ml); group 3, 100 ml stirred bioreactor cultureat low density (about 7×10⁵/ml). The culture and transplantationstrategies are summarized in Table 3. Mice were maintained aftertransplantation with insulin administration until glucose was measuredto be lower than 350 mg/dL, which is consistent with long-term survival.Blood glucose levels in mice after transplantation (FIG. 11 b) show thatbioreactor-cultured cells reversed diabetes in transplanted mice after aperiod of adaptation in vivo for over 3 weeks. Conversely, no micetransplanted with static-cultured cells showed sustained glucose levelsbelow the target of 350 mg/dL during the post-transplant period.Therefore, differentiated cells from stirred bioreactors provided highervolume cultures and supported higher cell densities; and high yield andquality of cells that can be generated in bioreactors were effective forreversing diabetes in mice.

TABLE 3 iPS-derived cell transplantation summary Group Mouse Cellstransplanted Transplanted site Group1 814  2.6 × 10⁶ Kidney capsule 10ml static 815 3.35 × 10⁶ Kidney capsule high density 816  2.7 × 10⁶Kidney capsule 818 2.25 × 10⁶ Kidney capsule Group 2 828  5.5 × 10⁶Kidney capsule 100 ml bioreactor 833 4.05 × 10⁶ Kidney capsule highdensity 820  8.2 × 10⁶ EFP 824 5.85 × 10⁶ EFP Group 3 827 3.45 × 10⁶Kidney capsule 100 ml bioreactor 819  4.1 × 10⁶ EFP low density

Discussion

Pluripotent stem cells are actively being explored for replacement ofdamaged tissues in many cell types. Although many cell types, such asneural, muscle, hematopoietic, bone and pancreas have been generatedfrom human pluripotent stem cells, clinical applications have laggedbehind in vitro studies, partly because of the limitation of large-scaleproduction of functional cells. Multiple mammalian cell lines such asHEK 293, CHO, Hela, NSO, vero cells have been cultured in bioreactorsfor producing recombinant proteins for clinic use. In industrialapplications, there are mainly two types of stirred large scale culturemethods. The first is a suspension culture in which cells grow in themedium and a stiffing mechanism drives the cells to keep them insuspension. The other option is an anchorage dependent culture in whichcells can either attach to the wall of the bioreactor, or attach to amicrocarrier bead, which is then suspended in the medium in a stirredbioreactor. Each of these methods has their advantages anddisadvantages, based on the cell line being used, as well as theproduction and scale. For clusters of cells such as EBs, the stirredbioreactor can be used, since beads are not required. Stirred suspensionbioreactor culture systems offer the regulation of multiple parameters,including O₂ and CO₂ tension, cytokine, glucose and serumconcentrations, as well as medium exchange rates, which may affect theviability and differentiation of stem cells into specific target cells.On the other hand, conventional stirrers may have the disadvantage ofgenerating shear forces, which can damage cells, like the early stagecells indicated in this study. Using a slow stiffing speed of 70 rpm,this study demonstrated that cell numbers and viability in stirredbioreactor cultures is comparable with static cultures. Further, staticcultures generated large clusters of EBs, which ultimately could impactgas and nutrient perfusion, causing cell necrosis.

Most of the previous published protocols for the differentiation ofhuman pluripotent stem cells into pancreatic cells use several basalmedium switches during the differentiation, and multiple cytokines areused to stimulate differentiation. Many of these protocols switch theculture from attachment at early stages to suspension cultures at laterstages, complicating scale-up and, increasing costs. Since therapeuticapplications require GMP manufacturing processes, reducing the number ofadded factors would facilitate clinical adaptation. In this study, weused only 3 cytokines, Activin A, Wnt3a and EGF and 1 monoclonalantibody, Anti-Shh before the cells were harvested for transplantation.Moreover, differentiation of iPS cells in this study was conducted withEBs (when cultured in suspension without antidifferentiation factors,some cells, such as stem cells, form three-dimensional multicellularaggregates called embryoid bodies (EBs)) throughout differentiation,which made scale up in stirred bioreactors easier.

The use of stirred bioreactors allowed the production of sufficientquantities of pancreatic progenitors to regulate glucose in vivo,without the loss of viability or reduction in growth rates. While the invitro analysis suggested that the products of the static and stirredbioreactor cultures were similar, the results in vivo indicate that onlythe cells from large-scale culture promoted normoglycemia in mice. Insummary, this study is the first to demonstrate that human iPS cells canbe differentiated in large-scale cultures to pancreatic progenitor cellsthat can reverse diabetes.

BIBLIOPGRAPHY

-   Cabrita G J M, et al. TRENDS in Biotechnology Vol. 21 No. 5 May    2003.-   Chu L and Robinson D K. Current Opinion in Biotechnology 2001,    12:180-187.-   D'Amour K A, et al. Nature Biotechnol. 2006; 24:1392-1401.-   Fridley K M, et al. TISSUE ENGINEERING: Part A Vol 00, No 00, 2010.-   Gerecht-Nir S, et al. Biotechnology and Bioengineering, vol. 86(5),    2004-   Jiang W, et al. Cell Res. 2007; 17:333-344.-   Krawetz R, et al. TISSUE ENGINEERING: Part C Vol 00, No 00, 2009.-   Kroon E, et al. Nat. Biotechnol. 2008; 26:443-452.-   Liew, C G. Rev Diabet Stud, 2010, 7(2):82-92.-   Maehr R, et al. Proc. Natl. Acad. Sci. U.S.A. 2009; 106:15768-15773.-   Phillips B W, et al. Stem Cells Dev. 2007; 16:561-578.-   Schroeder M, et al. Biotechnology and Bioengineering, vol. 92(7),    2005.-   Sen A, et al. Biotechnol. Prog. 2002, 18, 337-345.-   Shim J H, et al. Diabetologia 2007; 50:1228-1238.-   Takahashi K, et al. Cell 2007; 131:861-872.-   Takahashi K and Yamanaka S. Cell 2006; 126:663-676.-   Tateishi K, et al. J. Biol. Chem. 2008; 283:31601-31607.-   Yirme G, et al. Stem Cells and Development 17:1227-1242 (2008).-   Yu J, et al. Science 2007; 318:1917-1920.-   Zhang D W, et al. Cell Res. 2009; 19:429-438.-   Evans M J and Kaufman M H. Nature 292, 154-156 (1981).-   Martin G R. PNAS. 78, 7634-7638 (1981).-   Thomson J A, et al. Science 282, 1145-1147 (1998).-   Keller, G. et al. Gene & Dev. 19, 1129-1155 (2005).-   Soria B, et al. Diabetologia 44, 407-415 (2001).-   Kumar M and Melton D. Curr Opin Genet. Dev. 13, 401-407 (2003).-   Magliocca and Odorico. Curr Opin Organ Transplant 11, 88-93 (2006)-   Madsen, O. D. APMIS. 113, 858-875 (2006).-   D'Amour, K. A. et al. Nat. Biotechnol. 23, 1534-1541 (2005).-   Habener J. F., et al. Endocrinology 146, 1025-1034 (2005).-   Jensen J. Dev Dyn. 229, 176-200 (2004).-   Shi, Y. et al. Stem Cells 23, 656-662 (2005).

The complete disclosure of all patents, patent documents andpublications cited herein are incorporated herein by reference as ifindividually incorporated. The foregoing detailed description andexamples have been given for clarity of understanding only. Nounnecessary limitations are to be understood therefrom. The invention isnot limited to the exact details shown and described for variationsobvious to one skilled in the art will be included within the inventiondefined by the claims.

1. A method to differentiate stem cells to pancreatic progenitor cellscomprising the steps of: a) contacting the stem cells with at least onemember of the TGFβ family of cytokines and at least one member of theWnt family of proteins, b) contacting the cells obtained from step a)with at least one member of the TGFβ family of cytokines, at least onemember of the Wnt family of proteins, and an agent that inhibits sonichedgehog activity; and c) contacting the cells obtained from step b)with a member of the Epidermal growth factor (EGF) family of proteins;so as to yield pancreatic progenitor cells.
 2. The method of claim 1,wherein the at least one member of the TGFβ family of cytokines isactivinA or nodal.
 3. The method of claim 1, wherein the at least onemember of the Wnt family is Wnt3 or Wnt3A.
 4. The method of claim 1,wherein the at least one member of the EGF family is EGF.
 5. A method todifferentiate stem cells to pancreatic progenitor cells comprising thesteps of: a) contacting the stem cells with Activin A and Wnt3a, b)contacting the cells obtained from step a) with Activin-A, Wnt3a, and anagent that inhibits sonic hedgehog activity; and c) contacting the cellsobtained from step b) with EGF; so as to yield pancreatic progenitorcells.
 6. The method of claim 1, wherein the stem cells are embryonic oradult stem cells.
 7. The method of claim 1, wherein the stem cells areinduced pluripotent stem (iPS) cells.
 8. The method of claim 1 furthercomprising contacting the cells obtained from step c) with at least onemember of the TGFβ family of cytokines, at least one member of the Wntfamily of proteins, exendin4 or a combination thereof to yield cellsexpressing insulin.
 9. The method of claim 8, wherein the at least onemember of the TGFβ family of cytokines is GDF-11.
 10. The method ofclaim 8, wherein the at least one member of the Wnt family of proteinsis betacellulin.
 11. The method of claim 1, wherein the agent thatinhibits sonic hedgehog activity is cyclopamine or an anti-SHH antibody.12. The method of claim 8, wherein the cells expressing insulin orhaving increased expression of insulin secrete insulin, c-peptide or acombination thereof.
 13. The method of claim 8, wherein the insulin isinsulin-1.
 14. The method of claim 1, wherein the stem cells aremammalian cells.
 15. The method of claim 14, wherein the mammalian cellsare human cells.
 16. The method of claim 1, wherein the differentiationoccurs in a cell culture dish.
 17. The method of claim 1, wherein thedifferentiation occurs in bioreactor.
 18. A composition comprisingActivin-A, Wnt 3a and an agent that inhibits sonic hedgehog activity andstem cells.
 19. A composition comprising the cells prepared by themethod of claim 1 and cell culture medium or a pharmaceuticallyacceptable carrier.
 20. A method to prepare a composition comprisingcombining cells obtained by the method of claim 1 with cell culturemedium or a pharmaceutically acceptable carrier.
 21. A method to providepancreatic cells to a subject in need thereof comprising: a) contactingthe stem cells with at least one member of the TGFβ family of cytokinesand at least one member of the Wnt family of proteins, b) contacting thecells obtained from step a) with at least one member of the TGFβ familyof cytokines, at least one member of the Wnt family of proteins, and anagent that inhibits sonic hedgehog activity; and c) contacting the cellsobtained from step b) with a member of the Epidermal growth factor (EGF)family of proteins; and administering the cells so as to providepancreatic cells in the subject.
 22. The method of claim 21, wherein theat least one member of the TGFβ family of cytokines is activinA ornodal.
 23. The method of claim 21, wherein the at least one member ofthe Wnt family is Wnt3 or Wnt3A.
 24. The method of claim 21, wherein theat least one member of the EGF family is EGF.
 25. A method to providepancreatic cells to a subject in need thereof comprising: a) contactingstem cells with Activin A and Wnt 3a; b) contacting the cells obtainedfrom step a) with Activin-A, Wnt3a, and an agent that inhibits sonichedgehog activity; c) contacting the cells obtained from step b) withEGF; and administering the cells so as to provide pancreatic cells inthe subject.
 26. The method of claim 21, wherein the stem cells areembryonic or adult stem cells.
 27. The method of claim 21, wherein thestem cells are induced pluripotent stem (iPS) cells.
 28. The method ofclaim 21 further comprising contacting the cells obtained from step c)with at least one member of the TGFβ family of cytokines, at least onemember of the Wnt family of proteins, exendin4 or a combination thereofto yield cells expressing insulin prior to administration to thesubject.
 29. The method of claim 28, wherein the at least one member ofthe TGFβ family of cytokines is GDF-11.
 30. The method of claim 28,wherein the at least one member of the Wnt family or proteins isbetacellulin.
 31. A method to provide insulin expressing cells to asubject in need thereof comprising: a) contacting stem cells withActivin A and Wnt 3a; b) contacting the cells obtained from step a) withActivin-A, Wnt3a, and an agent that inhibits sonic hedgehog activity; c)contacting the cells obtained from step b) with EGF; d) contacting thecells obtained from step c) with GDF-11, betacellulin, exendin4 or acombination thereof so as to yield cells expressing insulin or havingincreased expression of insulin; and e) administering the cellsexpressing insulin or having increased expression of insulin to thesubject.
 32. The method of claim 21, wherein the stem cells areembryonic or adult stem cells.
 33. The method of claim 21, wherein thestem cells are induced pluripotent stem (iPS) cells.
 34. The method ofclaim 21, wherein the subject is a mammal.
 35. The method of claim 34,wherein the mammal is a human.
 36. The method of claim 21, wherein thesubject has a pancreatic disorder or injury.
 37. The method of claim 36,wherein the disorder comprises diabetes, obesity, pancreatic atresia,pancreas inflammation, alpha1-antitrypsin deficiency, hereditarypancreatitis, pancreatic cancer, pancreatic enzyme deficiency orhyperinsulinism.
 38. The method of claim 37, wherein the diabetes isType I or Type II diabetes.
 39. The method of claim 36, wherein theinjury is a result of physical trauma, chemical, radiation, aging,disease or combination thereof. 40-44. (canceled)